Carotenoid excited-state properties are characterized and compared in reaction centers (RCs) of wild-type (WT) Rhodobacter (Rb.) sphaeroides, and a mutant VR(L157), in which the near-infrared absorbance band associated with the primary electron donor, P, is missing. Energy transfer from the carotenoid (spheroidenone) S(2) and relaxed Si excited states to an adjacent monomeric-bacteriochlorophyll is unchanged between WT and the mutant RC samples. However, two other excited states, including a vibrationally hot SI state and a state referred to as S*, have distinct properties in the two RCs. The lifetime of the hot Si state is significantly shortened in the P-less mutant compared to WT RCs (450 fs vs 800 fs, respectively), and there is a nearly 2-fold decrease in the efficiency of energy transfer from the carotenoid to bacteriochlorophyll in the P-less mutant relative to WT RCs. The fact that both the observed hot S(1) excited state lifetime and the energy transfer efficiency decrease in the mutant implies that the intrinsic lifetime of the hot S(1) state in the P-less mutant has decreased. Interestingly, the S* state is observed only in the P-less mutant, and it is not present in the WT. The change in the hot SI lifetime between WT and mutant RCs, and the formation of the S* state only in the mutant, suggests that the carotenoid binding pocket in the P-less mutant is substantially altered. The excited-state behavior of spheroidene in WT RCs isolated from anaerobically grown cells was also characterized and compared with previous studies of spheroidene in the light-harvesting complex II (LH2) from Rb. sphaeroides. Differences in the photophysical properties of spheroidene between WT RCs and LH2 parallel those observed for spheroidenone between WT and VR(L157) mutant RCs. On the basis of the structural information available for both RCs and LH2, it appears that the hot S(1) state and the S* state are sensitive to the structural constraints imposed by protein carotenoid interactions. Finally, in the VR(L157) mutant, it is possible to directly observe the carotenoid triplet state, likely formed via quenching of the bacteriochlorophyll triplet state. This provides direct experimental evidence for triplet energy transfer to the carotenoid, a process that is integral to the photoprotective role of carotenoids in bacterial RCs.