Publisher Summary This chapter discusses the coupling factors from photosynthetic bacteria. Coupling factors (ATPase) have been isolated and purified from three genera of photosynthetic bacteria—namely, Rhodopseudomonas capsulata, Rhodospirillum rubrum (Rhodospirillaceae), and Chromatium vinosum (strain D) (Chromatiaceae). The first successful isolation and characterization of a coupling factor from a facultative photosynthetic bacterium was achieved for R. capsulata. Three methods have been reported for purification to homogeneity of the coupling factor from R. rubrum. The first procedure is based on the method first used for the partial purification of coupling factor from R. capsulate. The purification protocol includes three steps: extraction of an acetone powder of chromatophores with an ATP containing buffer, precipitation with ammonium sulfate between 30 and 55% saturation, and column chromatography on Sepharose 6B. Two additional steps have been introduced. The chapter also describes second procedure for purification to homogeneity of the coupling factor from R. rubrum. The main difference between the first and second method consists in the technique used for solubilizing the coupling factor from the membranes. A third procedure utilizes an affinity chromatography step for the final rapid stage of purification.