We have investigated the interaction of plastoquinol (PQH2), duroquinol (DQH2) and duroquinone (DQ) with the PQH2 oxidase site (Qc, Qo, or Qp site) of the chloroplast bf complex. By preilluminating thylakoids in the absence of artificial electron donors or acceptors, and under anaerobic conditions, we were able to probe the turnover of the bf complex with a fully reduced PQ pool. In the presence of NQNO to inhibit the reoxidation of cyt b, the half time for reduction of cyt b upon a single-turnover actinic flash was approximately 2-2.5 ms, with an initial rate of 252 sec-1. A second actinic flash given to this sample probed the initial rate of cyt b reduction with a partly (about 15-20%) oxidized PQ pool (DCMU was added in the dark to prevent reduction of the pool by PS2). The intial rate of cyt b reduction was nearly identical to that after the first actinic flash, indicating that this rate is most probably at or near the Vmax for the reaction (i.e. all Qo sites occupied by PQH2). However, when the PQ pool was fully reduced by addition of 0.1 mM DQH2, the half time increased to approximately 6 msec, with an initial rate of only 100 sec-1. Only at much higher concentrations of DQH2 did the initial rate of cyt b reduction approach that found in pre-illuminated samples. If the endogenous PQ was initially reduced by titration with Na dithionite, the initial rate of cyt b reduction was similar to that measured with the PQ pool reduced by preillumination. When DQH2 was introduced into samples titrated with dithionite, the rate of cyt b reduction remained rapid. The rate was about 90% of maximum after DQH2 was introduced into samples well purged of oxygen. We conclude that, in the presence of O2, a fraction of the added DQH2 was oxidized to DQ which acted as a competitive inhibitor of the Qo site. Starting with strictly anaerobic samples titrated with a series of DQ and DQH2 concentrations, where the PQH2 pool was at all times essentially completely reduced , we were able to demonstrate a competitive interaction at the Qo site among PQH2, DQH2 and DQ. By using an appropriate kinetic model-consisting of an enzyme (the bf complex), two alternate substrates (PQH2 and DQH2) and one competitive inhibitor (DQ)--we were able to simulate the kinetics of cyt b reduction that resulted from this competition. From these simulations, we concluded that the Vmax for oxidation of DQH2 and PQH2 were both about 250 sec-1. The predicted binding constants for the species at the Qo site depended on the assumed values of the partition coefficients of DQ and DQH2 into the thylakoid membrane. When the range of values for the partition coefficients suggested by Rich, et al () were input into the simulations, we were able to estimate lower bounds for binding constant of PQH2 (binding constant 2x10^5) to the site, and the relative binding constants of the other two species. We conclude that, with a completely reduced PQ pool, essentially all Qo sites were occupied with PQH2 indicating a fairly tight binding. By extrapolation from our data to zero added DQH2, approxiately 0.003 mM of added DQ is expected to displace PQH2 from half of the Qc sites. DQH2 can displace nearly all of the DQ from the sites, but only at high added concentrations. At the high concentrations of DQH2 typically employed in electron transfer assays-typically 0.5-1 mM-nearly all turnovers of the complex occur at the expense of DQH2. At lower concentrations, in the presence of O2, competitive inhibition by DQ can severely affect experimental results. Even under anaerobic conditions, a significant slowing of the initial rate of cyt b reduction was observed upon repetitive flashes, presumably because DQ produced in the repetitive flashes can compete for turnover. Care must be excersized when interpreting data obtained in the presence of DQ and DQH2. We have also found that decyl-ubiquinol (dUQH2) is a substrate for the bf complex, but with the product of partition coefficient into the membrane and binding consatnt into the Qo site greater than that of DQH2 and DQ.