Delayed fluorescence from isolated reaction centers of Rhodopseudomonas sphaeroides was measured to study the energetics of electron transfer from the bacteriochlorophyll complex ( P-870, or P) to the primary and secondary quinones (Q A and Q B). The analysis was based on the assumption that electron transfer between P and Q reaches equilibrium quickly after flash excitation, and stays in equilibrium during the lifetime of the P +Q − radical pair. Delayed fluorescence of 1Q reaction centers (reaction centers that contain only Q A) has a lifetime of about 0.1 s, which corresponds to the decay of P +Q − A. 2Q reaction centers (which contain both Q A and Q B) have a much weaker delayed fluorescence, with a lifetime that corresponds to that of P +Q − B (about 1 s). In the presence of o-phenanthroline, the delayed fluorescence of 2Q reaction centers becomes similar in intensity and decay kinetics to that of 1Q reaction centers. From comparisons of the intensities of the delayed fluorescence from P +Q − A and P +Q − B, the standard free energy difference between P +Q − A and P +Q − B is calculated to be 78 ± 8 meV. From a comparison of the intensity of the delayed fluorescence with that of prompt fluorescence, we calculate that P +Q − A is 0.86 ± 0.02 eV below the excited singlet state of P in free energy, or about 0.52 eV above the ground state PQ A. The temperature dependence of the delayed fluorescence indicates that P +Q − A is about 0.75 eV below the excited singlet state in enthalpy, or about 0.63 eV above the ground state.