In whole algae and isolated thylakoids, the maximum yield of Chl a fluorescence induced by a saturating single turnover flash is about half of the maximum yield observed under continuous illumination. The origins of this low fluorescence yield were investigated by measuring in fresh spinach thylakoids incubated under different conditions the fluorescence yield induced by a weak non-actinic flash fired alone ( F o) or 50 μs after a saturating laser flash ( F m) and also the PSII effective absorption cross-section ( σ PSII) derived from the flash energy saturation curves of F v ( F v = F m − F o). We observed that: (1) In the presence of a background blue light or after the chemical reduction of the primary quinone electron acceptor Q A, a saturating single turnover flash induced high fluorescence yields comparable to the maximum yield observed under continuous illumination. (2) Addition of carbonylcyanide- m-chlorophenylhydrazone (CCCP) in the assay medium increased the variable fluorescence ( F v = F m − F o) by 24% relative to the control and abolished its period-four oscillation under repetitive flashes. (3) After dark incubation of thylakoids under reducing conditions where most of the plastoquinone pool was chemically reduced, large increases of both F v σ PSII were observed. (4) In thylakoids treated with the inhibitor DCMU, both F o and F m were increased by 30% relative to the control but no significant change of σ PSII was observed. In contrast to intact thylakoids, F m increased significantly under repititive flashes in DCMU-treated thylakoids. Moreover, the enhancements of σ PSII following the plastoquinone chemical reduction were largely abolished in DCMU-treated thylakoids. From these observations, we conclude that although some limitations of the PSII donor side contribute to the low fluorescence yield after a single turnover flash, most of the fluorescence quenching present after a single turnover flash originates from the oxidized plastoquinone pool and/or from a unidentified component, possibly the putative quencher Q 2.