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The primary light-induced charge separation in reaction centers from Rhodopseudomonas sphaeroides R-26 has been investigated after excitation with laser pulses of 150 fsec duration within the longwave absorption band of the primary donor at 850 nm. An excited state of the primary donor, characterized by a broad absorption spectrum extending over the whole spectral range investigated (545-1240 nm), appeared within 100 fsec and gave rise to stimulated emission in the 870-to 1000-nm region with a 2.8-psec lifetime. The photooxidation of the primary donor, as measured at 1240 nm, and the photoreduction of the bacteriopheophytin acceptor, monitored at 545 nm and 675 nm, have been found to proceed simultaneously with a time constant of 2.8 ± 0.2 psec. Kinetics of absorbance changes at other probe wavelengths gave no indication that an accessory bacteriochlorophyll is involved as a transient electron acceptor.The mechanism by which light is converted into chemical free energy involves a sequence of very fast events between the absorption of a photon by the antenna pigments and the completion of the primary charge separation in the chlorophyll-protein complex, called the reaction center. Picosecond spectroscopy on reaction centers isolated from photosynthetic bacteria has revealed the presence of shortlived intermediates in this electron transfer process (1-11). However, the first steps take place in a time shorter than the duration of the pulses generally used and therefore have not been kinetically resolved. A previous study using subpicosecond pulses suggests that an electron has been transferred in -4 psec (5). However, the use of an excitation at 610 nm, which could directly excite one of the possible initial acceptors (bacteriochlorophyll), made it difficult to reach definite conclusions. In the present work, the earliest events in the charge separation process occurring in the reaction center of Rhodopseudomonas sphaeroides R-26 have been investigated by using a femtosecond spectroscopic technique, which associates a 100-fsec time resolution with tunability of the excitation in the near-infrared region (12, 13).Reaction centers from photosynthetic bacteria can be isolated in a functionally intact state and contain three polypeptides, four bacteriochlorophylls, two bacteriopheophytins, two quinones, and one non-heme iron atom (14). In the case of the reaction center from R. sphaeroides R-26, the main absorption bands of the pigments are located at 865, 800, 760, 590, and 540 nm. The 865-nm band, which bleaches upon (photo)oxidation of the reaction center, is ascribed to the primary donor (P). ENDOR data (15,16) indicate that in oxidized P (P+) the hole left by the removal of the electron is delocalized over a dimer of bacteriochlorophylls. Concomitant with the (photo)oxidation of P, the 800-nm band undergoes a change, which appears to be largely a blue shift. This shift is currently assigned to an electrochromic effect of P+ on the Qy transition of the two other bacteriochlorophyll molecules (called B) w...