Light-gradient photovoltage measurements in whole cells of the purple bacterium Rb. sphaeroides R26.1 were used to probe the trapping kinetics and the trapping efficiency resulting from 30 ps excitation at 532 nm. The time-course of the photovoltage is described by two distinct phases. The slower phase is characterized by a time constant of 190 ± 10 ps, contributing to about 60% of the total amplitude. It is independent of the excitation energy and of the initial fraction of reaction centers (RC) in the open state (primary donor reduced). This phase is assigned to the forward electron transfer from the bacteriopheophytin intermediary acceptor to the quinone. The fast phase is assigned to the appearance of the primary charge separated state (i.e., trapping). If all the RCs were in the open state before excitation, the time constant of the fast phase decreased from 55 ± 10 ps to less than 40 ps upon increasing the energy of the flash from less than 0.5 to more than 3 photons absorbed per RC. At the latter energy, 90 ± 5% of the RCs are closed by the flash. If the initial concentration of RCs in the closed state was increased from 0 to about 100%, either by background illumination or by preexcitation with another picosecond flash delivered 20 ns before the measuring flash, the mean trapping time increased from 55 ± 10 to 90 ± 15 ps for a probe flash energy corresponding to 0.5 photon absorbed per RC. The data are analyzed and described in terms of the competition between trapping and exciton-exciton annihilation in the lake model of the photosynthetic membrane of purple bacteria (Deprez, J., Paillotin, G., Dobek, A., Leibl, W., Trissl, H.-W. and Breton, J. (1990) Biochim. Biophys. Acta 1015, 295–303).