In isolated thylakoids, the differential effects of 2,5-dimethyl-p-benzoquinone (DMQ) and 2,6-dichloro-p-benzoquinonen (DCBQ) on chlorophyll a (Chl a) fluorescence transients and O₂ evolution data have been used to differentiate between active and inactive photosystem II (PSII) centers. This conclusion was challenged by Lavergne and Leci (Photosynth. Res., 35 (1993) 323–343). Thus we have systematically re-investigated this phenomenon using different concentrations of DMQ and DCBQ in thylakoids exposed to various light intensities. We show that the differential effects of DMQ and DCBQ on Chl a fluorescence transients in spinach thylakoids include a larger decrease in the variable Chl a fluorescence by DCBQ than by DMQ and a decrease in the F_o level with increasing [DCBQ] but not [DMQ] in the 5–150 μM range. These differential effects confirm the results of Lavergne and Leci, and thus sustain the conclusion that the effects of DMQ and DCBQ on Chl a fluorescence cannot be used to distinguish between active and inactive PSII centers. The data show that the decrease in Chl a fluorescence follows Stern-Volmer behavior. Furthermore, our data confirm that the rate of O₂ evolution is higher in the DCBQ-supported Hill reaction than in the DMQ-supported Hill reaction. These results can be explained by a more efficient ability of DCBQ to oxidize the plastoquinol pool or a more efficient ability of DCBQ to displace Q_B (secondary bound plastoquinone, a two-electron acceptor of PSII), rather than activating the inactive PSII centers. However, our results do not challenge the existence of inactive PSII.