The two-electron gate of Photosystem II (PSII) is known to function by transferring electrons from the reduced one electron acceptor Q - A (a bound plastosemiquinone) to the oxidized two-electron acceptor Q B (a bound plastoquinone), and then again from Q - A to the singly reduced Q - B , producing plastoquinol Q B H 2 . In this article, we have used three chloroacetates (monochloroacetate, MCA; dichloroacetate, DCA; and trichloroacetate, TCA), having different geometry and hydrophobicity, to probe the binding environment of the two-electron gate in spinach thylakoids. We first established that these chloroacetates up to 100 mM act specifically in the Q A Q B region by monitoring partial reactions of PSII as well as PSI, measuring thermoluminescence, specific for recombination reactions between the donor and acceptor sides of PSII, and studying chlorophyll (Chl) a fluorescence decay in the micro to millisecond region, specific for electron flow from Q - A to Q B (Q - B ). Further, the site of action was located on the D1-D2 protein through observations on the differential sensitivity of chloroacetates on specific D1-D2 mutants of the cyanobacterium Synechocystis sp. PCC 6803.Detailed measurements were then done to characterize the effect of chloroacetates on Q A Q B reactions. Data on the [Q - A ] decay kinetics led to the following observations: (1) chloroacetates (and acetate) not only increase the time constant of electron flow from Q - A to Q B (Q - B ), but increase the equilibrium [Q - A ] both after flash 1 and 2, and the degree of these effects (lowest to highest) is correlated with the geometry (increased number of chlorine moiety) and increased hydrophobicity of these inhibitors; the hierarchy is: acetate < TCA. (2) In comparison with flash 1, data after flash 2 (at pH 6) show relatively larger increases in [Q - A ] equilibrium with DCA and TCA. At pH 7.5, however, flash 1 effects were larger than flash 2 effects with all chloroacetates. (3) Bicarbonate reverses the inhibitory effect on Q - A to Q B (Q - B ) reactions also in a differential manner; the hierarchy for the most reversible (or least irreversible) is: acetate \>~ MCA > DCA > > TCA. (4) The pH dependence of the inhibitory effects on Q - A to Q B (Q - B ) are: the MCA and DCA effects are larger at pH 6 than at pH 7.5, but the TCA effects are higher at pH 7.5 than at pH 6. The above results, taken together with those in the literature, are in agreement with a picture of the Q A -Fe-Q B niche in the D1-D2 protein of PS II where quinones, herbicides, chloroacetates, formate as well as bicarbonate bind, but differently with different overlapping sites. Chloroacetates show effects on the two-electron gate that place them in-between the herbicides and the bicarbonate-reversible formate.