Inhibition of photosystem 2 (PS2) reducing side in pea chloroplasts by 10-* M DCMU [3-(3,4-dichlorophenyl)-l,l-dimethylurea], resulting from its binding at the secondary quinone acceptor site is manifested by suppression of millisecond time range delayed fluorescence (DF). In concentrations hi^er than 10-^ M DCMU binds to another site in PS2 and inhibits the oxidizing side, thus leading to suppression of DF with ti/2 = 2 s. Based upon DF measurements, DCMU-affinity of the second site was increased upon chaotropic action of oleic acid, while addition of polyethylene glycol that stabilizes polypeptides lead to preferential decrease in aífinity of the main binding site. The afFmities of the two sites differred also in their dependence upon pH of the mediiun. Thus both binding sites can háve high or low aíiitities to DCMU depending on the statě of their microenvironment. A lower affmity to DCMU of the site responsible for inhibition of the oxidizing side is probably related to its more rigid conformation.