Effects of the alleochemical sorgoleone on photosynthetic electron transport by oxygen-evolving chloroplast thylakoids and Triton X-100-prepared Photosystem II (PSII) membranes were analyzed. The Hill activity of the thylakoids proved to be at least as sensitive to inhibition by sorgoleone as it was to DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea], a potent herbicidal inhibitor of PSII. However, a Photosystem I (PSI) partial reaction was not affected by a 10-fold greater concentration of sorgoleone than is required for complete inhibition of Hill activity. Measurements of flash-induced chlorophyll a variable fluorescence showed that sorgoleone neither dissipated excitation energy nor diminished the amplitude of chlorophyll a variable fluorescence. However, it inhibited the decay of variable fluorescence as effectively as DCMU, which blocks the oxidation of the PSII-reduced primary electron acceptor, Q- A, by the PSII secondary electron acceptor, QB, by displacing QB from the D1 protein. Additionally, sorgoleone competitively inhibited the binding of [14C]atrazine to the QB locus. Increasing durations of trypsin proteolysis of the PSII membranes or thylakoids and of the QB-binding niche itself caused parallel losses of inhibition of O2 evolution from sorgoleone and DCMU, as well as from bromoxynil, a phenol-type herbicide also binding to the QB locus.