The long-lived, light-induced radical Y(D) of the Tyr161 residue in the D2 protein of Photosystem II (PSII) is known to magnetically interact with the CaMn(4) cluster, situated approximately 30 A away. In this study we report a transient step-change increase in Y(D) EPR intensity upon the application of a single laser flash to S(1) state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of Y(D) by the OEC in the S(2) state, as a consequence of the single laser flash turnover. The post-flash decay reflected S(2)-->S(1) back-turnover, as confirmed by their correlations with independent measurements of S(2) multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using Y(D) as a probe.