It has been shown previously that ATP is released into extracellular space from pre- and postsynaptic sources in peripheral synapses. The extracellular metabolism of ATP is likely to affect nucleotide- and nucleoside-mediated regulation of neurotransmission. The enzymes responsible for ATP breakdown are nucleotidases whose active site faces the extracellular space. ATPase and ADPase Ca(2+)-dependent activities were characterized in presynaptic plasma membrane preparation from the electric organ of Torpedo. Features described were in accordance with the presence of an ATP-diphosphohydrolase (apyrase EC 3.6.1.5) in this fraction. Active site studies using the affinity label 5'-fluorosulfonylbenzoyladenosine were performed on Torpedo apyrase. ATPase and ADPase Ca(2+)-dependent activities were inhibited with 5'-fluorosulfonylbenzoyladenosine. From this study it is concluded: (1) 5'-fluorosulfonylbenzoyladenosine binds specifically to the active site of apyrase. (2) Divalent cations accelerate the apyrase inactivation rate. (3) Divalent cations are not required for the binding of either the substrate or the inhibitor to the active site. (4) The apyrase active site is more specific for highly phosphorylated nucleotides. The results presented may be extrapolated to apyrases from other sources. The importance of this enzyme and its regulation are discussed.