Abstract Measurements of the area bounded by the variable fluorescence induction curve and the maximum fluorescence yield as a function of redox potential led I. Ikegami and S. Katoh ((1973) Plant Cell Physiol. 14, 829–836) to propose the existence of a high-potential electron acceptor, Q 400 ( E m7.8 = 360 mV), associated with Photosystem II (PS II). We have generated the oxidized form of this acceptor (Q + 400) using ferricyanide and other oxidants in thylakoid membranes isolated from a mutant of Chlamydomonas reinhardtii lacking Photosystem I and the cytochrome b 6 f complex. Q + 400 was detected by a decrease in the extent of reduction of the primary quinone electron acceptor, Q A, in a low-intensity light flash exciting PS II reaction centers only once. EPR measurements in the presence of Q + 400 indicated the presence of new signals at g = 8, 6.4 and 5.5. These disappeared upon illumination at 200 K or upon reduction with ascorbate. Mössbauer absorption attributed to the Fe 2+ of the Q A-Fe 2+ acceptor complex of PS II disappeared upon addition of ferricyanide due to the formation of Fe 3+. The Fe 2+ signal was restored by subsequent addition of ascorbate. All of these spectroscopic signals show similar pH-dependent ( n = 1) midpoint potentials (approx. −60 mV / pH unit) and an E m7.5 = 370 mV. We assign the EPR signals to the Fe 3+ state of the quinone-iron acceptor. Electron transfer to the Fe 3+ is responsible for the decrease in Q A reduction upon single-hit flash excitation. The properties of the Fe 3+ Fe 2+ redox couple are consistent with those of Q + 400 Q 400 and we conclude that the iron of the Q A-Fe acceptor complex is responsible for this species.