In the presence of acetone and an excess of exogenous plant pheophytins, bacteriopheophytins in the reaction centers from Rhodobacter sphaeroides RS601 were replaced by pheophytins at sites H(A) and H(B), when incubated at 43.5 degrees C for more than 15 min. The substitution of bacteriopheophytins in the reaction centers was 50% and 71% with incubation of 15 and 60 min, respectively. In the absorption spectra of pheophytin-replaced reaction centers (Phe RCs), bands assigned to the transition moments Q(X) (537 nm) and Q(Y) (758 nm) of bacteriopheophytin disappeared, and three distinct bands assigned to the transition moments Q(X) (509/542 nm) and Q(Y) (674 nm) of pheophytin appeared instead. Compared to that of the control reaction centers, the photochemical activities of Phe RCs are 78% and 71% of control, with the incubation time of 15 and 60 min. Differences might exist between the redox properties of Phe RC and of native reaction centers, but the substitution is significant, and the new system is available for further studies.