The kinetics of recombination of the P+HA- radical pair were compared in wild-type reaction centers from Rhodobacter sphaeroides and in seven mutants in which the free energy gap, Delta G, between the charge separated states P+BA- and P+HA- was either increased or decreased. Five of the mutant RCs had been described previously, and X-ray crystal structures of two newly constructed complexes were determined by X-ray crystallography. The charge recombination reaction was accelerated in all mutants with a smaller Delta G than in the wild-type, and was slowed in a mutant having a larger Delta G. The free energy difference between the state P+HA- and the PHA ground state was unaffected by most of these mutations. These observations were consistent with a model in which the P+HA- -> PHA charge recombination is thermally activated and occurs via the intermediate state P+BA-, with a mean rate related to the size of the Delta G between the states P+BA- and P+HA- and not the Delta G between P+HA- and the ground state. A more detailed analysis of charge recombination in the mutants showed that the kinetics of.:he reaction were multiexponential, and characterized by similar to 0.5, similar to 1-3, and 7-17 ns lifetimes, similar to those measured for wild-type reaction centers. The exact lifetimes and relative amplitudes of the three components were strongly modulated by the mutations. Two models were considered in order to explain the observed multiexponentiality and modulation, involving heterogeneity or relaxation of P+HA- states, with the latter model giving a better fit to the experimental results.