Delayed fluorescence from Rhodopseudomonas sphaeroides chromatophores was studied with the use of short flashes for excitation. Although the delayed fluorescence probably arises from a back-reaction between the oxidized reaction center bacteriochlorophyll complex (P+) and the reduced electron acceptor (X-), the decay of delayed fluorescence after a flash is much faster (tau1/2 approximately 120 mus) than the decay of P+X-. The rapid decay of delayed fluorescence is not due to the uptake of a proton from the solution, nor to a change in membrane potential. It correlates with small optical absorbance changes at 450 and 770 nm which could reflect a change in the state of X-. The intensity of the delayed fluorescence is 11-18-fold greater if the excitation flashes are spaced 2 s apart than it is if they are 30 s apart. The enhancement of delayed fluorescence at high flash repetition rates occurs only at redox potentials which are low enough (less than +240 mV) so that electron donors are available to reduce P+X- to PX- in part of the reaction center population. The enhancement decays between flashes as PX- is reoxidized to PX, as measured by the recovery of photochemical activity. Evidently, the reduction of P+X- to PX- leads to the storage of free energy that can be used on a subsequent flash to promote delayed fluorescence. The reduction of P+X- also is associated with a carotenoid spectral shift which decays as PX- is reoxidized to PX. Although this suggests that the free energy which supports the delayed fluorescence might be stored as a membrane potential, the ionophore gramicidin D only partially inhibits the enhancement of delayed fluorescence. With widely separated flashes, gramicidin has no effect on delayed fluorescence. At redox potentials low enough to keep X fully reduced, delayed fluorescence of the type described above does not occur, but one can detect weak luminescence which probably is due to phosphorescence of a protoporphyrin.