Forward electron transfer at room temperature from the secondary acceptor A1 (phylloquinone) to the iron-sulfur centers F(X), F(B), and F(A) 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., & Setif, 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 almost-equal-to 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 almost-equal-to 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 F(A) and F(B), only the 25-ns phase is conserved and the absorbance changes remaining after its completion decay with t1/2 almost-equal-to 250 mus. It is concluded that the 25-ns phase reflects electron transfer from A1- to F(X) in approximately 65% of the centers, whereas the remaining 35% of A1- is reoxidized with t1/2 almost-equal-to 150 ns under moderate redox conditions. The deviations between the spectra of the two phases can be explained with the assumption that electron transfer from F(X-) to (F(A), F(B)) also proceeds with t1/2 almost-equal-to 150 ns and contributes significantly to the total spectrum of the 150-ns phase, implying that the F(X-)/F(X) difference spectrum deviates from the (F(A),F(B))-/(F(A),F(B)) Spectrum. Possible kinetic schemes for forward electron transfer in PSI-beta particles are discussed; assuming that the 25-ns phase reflects the establishment of a redox equilibrium between reduced A1 and F(X), the redox potentials of A1 and F(X) are found to be very close. Different types of PSI particles from spinach, which were subjected to less harsh preparation procedures, also exhibit a biphasic reoxidation of A1- but smaller relative amplitudes of the 25-ns phase, down to only 30% for a sample prepared without detergent. It is suggested that PSI in native spinach membranes could behave similarly to the cyanobacterial PSI particles.