Femtosecond transient absorbance spectra of quinone-depleted Rhodobacter sphaeroides R-26 reaction centers in the Q(X) transition region have been measured at 15 K under various excitation conditions. This study focuses on the excitation wavelength dependence and excitation intensity dependence of the formation of charge-separated states on the A- and B-side of the reaction center, judging from the bleaching of the 533 nm (B-side) and 544 nm (A-side) ground-state transitions of the reaction center bacteriopheophytins (HA and HB) Upon low-intensity selective excitation directly into the bacteriopheophytin Q(X) transitions (near 760 nm), bleaching of both ground-state bacteriopheophytin Q(X) transitions (533 and 544 nm) appeared immediately, showing that initially either the A- or B-side bacteriopheophytin could be excited. However, both excited states ultimately resulted in P+HA- formation under these conditions. Low-intensity excitation at any of the various wavelengths (595, 750, 760, and 880 nm) showed no difference in the kinetics of the A-side charge separation forming P+HA- and no substantial formation of the B-side charge-separated state, P+HB-. In contrast, high-intensity 595 nm excitation resulted in substantial long-lived bleaching of the B-side bacteriopheophytin ground-state transition at 533 nm. This 533 nm bleaching was formed with essentially the same time constant as the bleaching at 544 nm due to A-side charge separation.;Both bleaching bands persisted at the longest times measured (hundreds of picoseconds) in quinone-removed reaction centers. The long-lived bleaching at 533 nm using high-intensity excitation most likely represents the formation of P+HB- with a relative yield (percent of total charge separate state) of nearly 40%. One possible mechanism for B-side electron transfer is that two-photon excitation of the reaction center resulting in the state P*B-B* makes P+BB-thermodynamically accessible.