Photosystem I (PSI) is a large pigment-protein complex that unites a reaction center (RC) at the core with approximately 100 core antenna chlorophylls surrounding it. The RC is composed of two cofactor branches related by a pseudo-C2 symmetry axis. The ultimate electron donor, P(700) (a pair of chlorophylls), and the tertiary acceptor, F(X) (a Fe(4)S(4) cluster), are both located on this axis, while each of the two branches is made up of a pair of chlorophylls (ec2 and ec3) and a phylloquinone (PhQ). Based on the observed biphasic reduction of F(X), it has been suggested that both branches in PSI are competent for electron transfer (ET), but the nature and rate of the initial electron transfer steps have not been established. We report an ultrafast transient absorption study of Chlamydomonas reinhardtii mutants in which specific amino acids donating H-bonds to the 13(1)-keto oxygen of either ec3(A) (PsaA-Tyr696) or ec3(B) (PsaB-Tyr676) are converted to Phe, thus breaking the H-bond to a specific ec3 cofactor. We find that the rate of primary charge separation (CS) is lowered in both mutants, providing direct evidence that the primary ET event can be initiated independently in each branch. Furthermore, the data provide further support for the previously published model in which the initial CS event occurs within an ec2/ec3 pair, generating a primary ec2(+)ec3(-) radical pair, followed by rapid reduction by P(700) in the second ET step. A unique kinetic modeling approach allows estimation of the individual ET rates within the two cofactor branches.