The triplet state of the carotenoid spheroidene. a pigment cofactor of reaction centers (RCs) from the photosynthetic bacterium Rhodobacter sphaeroides 2.4.1., was studied with time-resolved direct-detection EPR under polarized light excitation (magnetophotoselection). Four types of magnetophotoselection experiments, differing by excitation wavelength and temperature. enabled determination of the orientations of the optical transition moments of the reaction center spheroidene and the primary donor relative to the principal triplet axes of these molecules. Making use of the projections of the primary donor principal triplet axes onto the X-ray structural coordinate system determined earlier, we were able to calculate for the first time the orientation of the spheroidene optical transition moment and its principal triplet axes relative to the RC molecular frame. The same type of experiment on carotenoidless reaction centers from Rb. sphaeroides R26 reconstituted with spheroidene demonstrated a significant difference between the native and reconstituted molecules with regard to the orientation of the optical transition moment of the carotenoid relative to its triplet principal axes frame.