N6-O- and p-fluorobenzoyladenosine 5'-triphosphates (IIIc and IIc, respectively) have been synthesized as potential adenosine 5'-triphosphate (ATP) site-directed reagents for enzymes. IIc and IIIc were substrates of yeast hexokinase; neither they nor the corresponding ADP derivatives inactivated yeast hexokinase or rabbit pyruvate kinase. IIc rapidly inactivated rabbit and carp muscle adenylate kinases; the effect is probably ATP site directed because N6-benzoyl-ATP did not inactivate and was a substrate (Vmax = 28 and 10%, respectively, that of ATP), and because of ATP retarded the inactivation. The inactivations followed pseudo-firsr-order kinetics; in the presence of 2.64 mM ATP at 0 degrees the half-life of the rabbit kinase was 210 min with 50 muM IIc and the half-life of the carp kinase was 130 min with 100 muM IIc. Adenylate kinase of pig muscle was inactivated by IIc in a manner similar to the rabbit and carp enzymes except that the rate of inactivation exhibited an inflexion. IIIc inactivated rabbit, pig, and carp adenylate kinases by pseudo-first-order kinetics; the rate constants for inactivation at 0 degrees were 9.1 X 10(-3), 1.3 X 10(-3), and 1.9 X 10(-3) min-1 and the apparent dissociation constants (K) of the IIIc-enzyme complexes were 710, 970, and 720 muM, respectively. From the substrate properties of IIIc alone and in admixture with ATP its dissociation constants (Ki) from the ATP sites of the enzymes were found to be 500, 700, and 845 muM, respectively. The similarity between the K and Ki values, together with marked retardation of the inactivations by ATP, indicates that IIIc is an ATP-site-directed reagent for the three adenylate kinases.