Forward electron transfer at room temperature from the secondary acceptor A1 (phylloquinone) to the iron-sulfur centers FX, FB, and FA was studied by flash-absorbance spectroscopy in different photosystem I (PSI) preparations in order to resolve the controversy concerning the kinetics of A1-reoxidation during forward electron transfer [half times of 15 ns [Mathis, P., & Sétif, P. (1988) FEBS Lett. 237, 65-68] and 200 ns [Brettel, K. (1988) FEBS Lett. 239, 93-98] were reported for PSI particles from spinach and Synechococcus sp., respectively]. The monophasic kinetics with t1/2 approximately 200 ns could be reproduced with PSI particles from another cyanobacterium (Synechocystis sp. PCC 6803). In so-called PSI-beta particles from spinach, containing all membrane-bound electron carriers and approximately 65 antenna chlorophylls per reaction center, the flash-induced absorbance increase around 370 nm, which is indicative of the formation of A1-, decays biphasically with t1/2 approximately 25 and 150 ns and relative amplitudes of approximately 65 and 35%, respectively. The difference spectra of these two phases were determined between 330 and 500 nm; they agree well below 380 nm but deviate significantly at higher wavelengths. The spectrum of the sum of the two phases is similar to the spectrum of the 200-ns phase in cyanobacteria. Upon chemical reduction of the terminal acceptors FA and FB, only the 25-ns phase is conserved and the absorbance changes remaining after its completion decay with t1/2 approximately 250 microseconds.(ABSTRACT TRUNCATED AT 250 WORDS)