Relative distances between the the primary donor P, the intermediary pheophytin acceptor H, and the iron-quinone acceptor Q of bacterial reaction centers were determined by recording laser flash-induced photovoltages in two experimental systems with nanosecond time resolution. In one system a suspension of chromatophores was subjected to a light gradient and in the other system chromatophores were spread at a heptane/water interface. The 10-ns back reaction occurring in reaction centers with reduced Q could be time resolved. The initial photovoltage amplitude under conditions in which the charge separation proceeded up to the state [P(+)H(-)] was about (2/3) of that when it proceeded up to the state [P(+)HQ(-)]. If the amplitude of the photovoltage is considered to be proportional to the spatial displacement of charges, this result means that pheophytin lies closer to Q than to P.