The electron transfer in photosystem I (PS I) from the secondary acceptor A(1) to the iron-sulfur centers is studied by X-band transient EPR with a time resolution of similar to 50 ns. Results are presented for a series of different PS I preparations from the cyanobacterium Synechococcus 6301 ranging from whole cells to core particles in which the iron-sulfur centers have been successively removed. In addition, results from PS I preparations from spinach and the cyanobacterium Synechocystis 6803 are presented. In all samples containing iran-sulfur centers, two consecutive spin-polarized EPR spectra are observed. The two signals have previously been assigned to the charge-separated states P(700)(+.)A(1)(-.) and P-700(+.)(FeS)(-), where (FeS) is one of the three iron-sulfur centers, F-X, F-A, or F-B [Bock, C., van der Est, A., Brettel, K., & Stehlik, D. (1989) FEBS Lett. 247, 91-96]. In agreement with this, the second spectrum is not observed in the sample in which the iron-sulfur centers have been removed. For (P-700-F-X), core particles which do not contain F-A and F-B, the second spectrum can unambiguously be assigned to the pair (P700FX-)-F-+.. In all samples containing F-X, the transition from the first to the second spectrum occurs with t(1/e) approximate to 280 ns (t(1/2) approximate to 190 ns) both in the presence and absence of F-A and F-B, which strongly suggests that this phase reflects electron transfer from A(1)(-.) to F-X in intact PS I. For the spinach particles and for the cyanobacterial (P-700-F-X) core particles, the EPR data show evidence for the presence of a fraction of the reaction centers in which electron transfer from A(1)(-.) to F-X is faster than the response time of the spectrometer, in agreement with optical data for the same samples. It is shown that when this fraction is taken into account the early spectrum extracted from the EPR data sets shows no net spin polarization and is identical for all samples except the (P-700-A(1)) core particles. Possible reasons for the differences in this sample are discussed.