In Rhodopseudomonas sphaeroides, light causes the transfer of electrons from bacteriochlorophyll to ubiquinone in the photochemical reaction centers. Electrons from this “primary” ubiquinone move on to a secondary ubiquinone. We have studied the manner in which o-phenanthroline inhibits the transfer of electrons from primary to secondary ubiquinone in chromatophores (intracytoplasmic membrane fragments) and isolated reaction centers of Rp. sphaeroides. The formation of anionic semiquinones, both primary and secondary, is signaled by an absorption band at 450 nm and by band shifts of bacteriopheophytin and bacteriochlorophyll in the near infrared. The pattern of band shifts is different for primary and secondary semiquinone, allowing us to distinguish which quinone has become semireduced. This point was established for isolated reaction centers [Vermeglio, A. & Clayton, R. K. (1977) Biochim. Biophys. Acta 461, 159-165] and is confirmed here for chromatophores. By adding o-phenanthroline at various times during a sequence of actinic light flashes and monitoring the resulting optical absorbance changes, we have found that o-phenanthroline inhibits electron transfer from primary semiquinone to secondary quinone if the latter is in its oxidized form, but not if the latter is semireduced. Our findings can explain the decay kinetics of delayed fluorescence from bacteriochlorophyll in Rp. sphaeroides as measured by R. P. Carithers and W. W. Parson [(1976) Biochim. Biophys. Acta 440, 215-232].