Singlet oxygen (¹O₂) scavenging activity of plastoquinol in photosystem II (PSII) of higher plants was studied by electron paramagnetic resonance (EPR) spin-trapping technique. It is demonstrated here that illumination of spinach PSII membranes deprived of intrinsic plastoquinone results in ¹O₂ formation, as monitored by TEMPONE EPR signal. Interestingly, the addition of exogenous plastoquinol (PQH₂-1) to PQ-depleted PSII membranes significantly suppressed TEMPONE EPR signal. The presence of exogenous plastoquinols with a different side-chain length (PQH₂-n, n isoprenoid units in the side chain) caused a similar extent of ¹O₂ scavenging activity. These observations reveal that plastoquinol exogenously added to PQ-depleted PSII membranes serves as efficient scavenger of ¹O₂.