Ecto-apyrase is a widespread enzymatic activity that hydrolyses tri- and diphosphonucleotides and consequently controls the amount of available extracellular ATP and ADP. In the nervous system, purines have important neuromodulatory actions, acting at pre- and postsynaptic sites, and consequently, ecto-apyrase may play an indirect role in the modulation of nucleotide- and nucleoside-mediated processes. The azido-nucleotides have been largely employed to characterize the nucleotide binding sites of several proteins. In the present work the azido-nucleotides are described as putative substrates for apyrase activity in a presynaptic plasma membrane preparation (PSPM) from the Torpedo electric organ. Both 8-N3-ATP and 8-N3-ADP were hydrolyzed in a calcium-dependent manner showing Vmax of 23.8 +/- 4.8 and 14.5 +/- 3 U/mg of protein, and Km values (in microM) of 116 +/- 39 and 119 +/- 4, respectively. Vmax for calcium-dependent hydrolysis of ATP and ADP were significantly higher: 59.2 +/- 3.9 and 32.9 +/- 3.5 U/mg of protein respectively, while Km values did not show any significant differences regarding azido-nucleotides: 83.8 +/- 12 microM for Ca2+-ATP and 121 +/- 34 microM for Ca2+-ADP. The photoactivation of the PSPM in the presence of the azido-derivatives results in an irreversible inactivation of apyrase activity, showing an IC50 of 10 microM and a maximal inhibitory effect of 38 and 60% on Ca2+-ATPase and Ca2+-ADPase activities. Apyrase was protected from inactivation by nucleotides that are natural substrates for this enzymatic activity and also by AMP while adenosine did not protect from apyrase inhibition.