The reaction center of Rhodopseudomonas sphaeroides is an integral membrane protein complex responsible for primary photochemical charge separation in photosynthesis. We report the synthesis of two of the three subunits of the photosynthetic reaction center using a DNA-directed in vitro transcription-translation system prepared from R. sphaeroides. The in vitro-synthesized polypeptides, as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had apparent Mrs of 24,000 and 21,000 and were shown to be synthesized in equimolar amounts. This corresponds precisely to the in vivo reaction center subunits M and L, respectively. The in vitro-synthesized polypeptides were immunoprecipitated with antibody prepared against whole native reaction centers. In addition, the identity of the in vitro-synthesized polypeptides as L and M was verified by comparing the protease digestion products of in vivo- with in vitro-synthesized reaction center subunits. Both of the in vitro-synthesized polypeptides were also found to partition with the particulate material in the transcription-translation system and to associate with added membranes.