Ammonium chloride, an uncoupler of photophosphorylation which stimulates the membrane-bound chloroplast coupling factor ATPase when added after light/dithiothreitol activation, causes a decrease in the number of extra water oxygens incorporated into the phosphate formed during ATP hydrolysis. This observation is in contrast to the long-reported insensitivity of intermediate Pi:H2O oxygen exchange to uncoupler dinitrophenol in the mitochondrial F1 ATPase system. The effect of ammonium chloride on the CF1-catalyzed oxygen exchange reaction is consistent with ATPase activity stimulation caused by increased partitioning forward of the enzyme . products complex. In line with the oxygen exchange data, ammonium chloride causes an increase in the apparent Km of the enzyme for substrate ATP. The effect of ammonium chloride on the pattern of the intermediate Pi:H2O oxygen exchange is not a threshold phenomenon; the extent of exchange decreases in a continuous fashion, paralleling the stimulation of ATPase activity. The uncoupler CF3OPhzC(CN)2 also decreases the extent of oxygen exchange upon stimulating the membrane-bound ATPase, while phlorizin, an energy-transfer inhibitor, has essentially no effect on exchange although it inhibits the ATPase reaction. Similar to the effect of chemical uncoupling on the membrane-bound enzyme, physical removal of the coupling factor ATPase from the thylakoid membrane also results in an increase in forward partitioning of the enzyme . ADP . Pi complex. The modulation of oxygen exchange observed by altering the degree of coupling is similar to that which accompanies changing ATP concentration in the mitochondrial ATPase system [Russo, J. A., Lamos, C. M. and Mitchell, R. A. (1978) Biochemistry 17,473-480 and Choate, G. L., Hutton, R. L. and Boyer, P. D. (1979) J. Biol. Chem. 254, 286-290]. However, the uncoupler modulation is not readily correlated with the degree to which multiple catalytic sites are occupied by substrate.