A key step of photosynthetic solar energy conversion involves rapid light-induced sequential electron-transfer steps that result in the formation of a stabilized charge-separated state. These primary reactions take place in large integral membrane reaction center (RC) proteins, wherein a series of donor/acceptor cofactors are specifically positioned for efficient electron transfer. RCs can be divided in two classes, Type I and Type II and examples of both types, photosystem I (PS I) and photosystem II (PS II), are involved in the oxygenic photosynthesis of higher plants, cyanobacteria, and algae. High-resolution X-ray crystal structures reveal that PS I and PS II contain two nearly symmetric branches of redox cofactors, termed the A and B branches. While unidirectional ET along the A branch in Type II RCs is well established, there is still a debate of whether primary photochemistry in Type I RCs is unidirectional along the A branch or bidirectional proceeding down both of the A and B branches. Light-induced electron transfer through the B branch has been observed in genetically modified PS I and in native PS I pretreated with strong reducing conditions to reduce three [4Fe-4S] clusters, the terminal electron acceptors of PS I; however, the extent of asymmetry of ET along both cofactor branches remains an open question. To prove that bidirectional ET in PS I is not simply an artifact of a reducing environment or genetic modification and to determine the degree of PS I ET asymmetry, we have examined biochemically modified Synechococcus leopoliensis PS I RCs, wherein the [4Fe-4S] clusters F-X, F-A, and F-B have been removed to prevent secondary ET from phylloquinones (A(1A)/A(1B)) to F-X. For these Fe-removed proteins, we observe that ET along both the A and B branches occurs with a ratio close to 1. Together with previously reported data, the concomitant structural and kinetic information obtained with HF EPR unambiguously proves the bidirectional nature of ET in PS I over a broad temperature range.