The arginine-binding reagents, butanedione and phenylglyoxal, inhibit the ATPase and the ATP-P, exchange activities of an ATP synthetase preparation (complex V) isolated from bovine heart mitochondria. The second order rate constants for inhibitor-enzyme interaction derived from the analysis of inhibition kinetics of ATPase and ATP-P, exchange activities are, respectively, 6.4 and 15.4 M-’ min-I for butanedione as inhibitor and 36.8 and 69.2 M-’ min-’ for phenylglyoxal as inhibitor. These data indicate that phenylglyoxal is the more effective inhibitor and ATP-P, exchange is the more susceptible activity. The estimated reaction orders with respect to inhibitor concentration varied between 0.77 and 1.15, thus suggesting an approximately equimolar inhibitor-enzyme interaction for inhibition of either ATP hydrolysis or ATP-P, exchange. The inhibition of ATPase activity by phenylglyoxal appeared to be noncompetitive with respect to ATP. Data for ATP-P, exchange could not be similarly analyzed because of substrate inhibition at high ATP concentrations, which became more effective in the presence of phenylglyoxal. ADP protects against phenylglyoxal inhibition of the ATPase activities of complex V and soluble ATPase (F,) from mitochondria, and as tested with complex V the degree of protection is a function of ADP concentration. Little protection is afforded by ADP against phenylglyoxal inhibition of complex V ATP-P, exchange activity. In complex V, both activities are protected, however, by 2,4-dinitrophenol, while no protective effect of the uncoupler was observed on the inhibition of ATPase activity of soluble F,. In submitochondrial particles butanedione inhibited the equilibrium binding of the uncoupler 2-azido-4nitrophenol to the mitochondrial uncoupler-binding site. The above results are consistent with the possible presence in F, (ATPase) of an essential arginyl residue involved in ATPase activity and a second and more susceptible argi