Publisher Summary The membrane fragments prepared from a variety of photosynthetic bacteria, when illuminated, can synthesize adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (P i ). Chromatophores prepared from the facultative photoheterotroph, Rhodospirillum rubrum , catalyze the hydrolysis of ATP into ADP and P i (ATPase activity); this activity is correlated with the reversibility of the energy conservation system leading to ATP formation. Rhodospirillum rubrum cells are grown in 1-liter flat bottles at 30 ° C under continuous illumination (approximately 1000 foot-candles) from a bank of tungsten lamps. ATP labeled in the terminal phosphate group is synthesized by means of the ATP–P i exchange reaction by illuminated chromatophores. The assay is based on the measurement of 32 P i liberated from γ- 32 P–ATP. A cell suspension of the blue-green mutant strain (G-9) of R. rubrum (80-mg/ml wet weight) is disrupted by sonication and fractionated. Protein is determined by the biuret method; bacteriochlorophyll is estimated spectrophotometrically. The rate of ATPase activity is maximal at pH 7.5; activities at pH 7 and pH 8 are 85% and 95%, respectively, of that at pH 7.5. The activity stimulated by 2,4-dinitrophenol (DNP) is also maximal at pH 7.5.