The release of ATPase activity evoked by electrical field stimulation (EFS) (8 Hz, 25 s) was investigated in several tissues in which adenosine 5'-triphosphate (ATP) acts as a neurotransmitter. Superfusate collected during EFS of sympathetic nerves of the guinea-pig, rat and mouse isolated vas deferens and parasympathetic nerves of the guinea-pig isolated urinary bladder contained ATPase activity. ATP breakdown was fastest in superfusate collected from the guinea-pig isolated vas deferens. However, EFS of the enteric nerves of the guinea-pig isolated taenia coli did not release any detectable ATPase. The ATPase released from the guinea-pig isolated vas deferens metabolized ATP at similar rates at incubation temperatures of 37 degrees C and 20 degrees C. Lineweaver-Burke analysis of the initial rates of ATP hydrolysis gave a K(M) of 39 microM and a V(max) of 1039 pmol ATP metabolized min(-1) ml(-1) superfusate. 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL 67156), pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and pyridoxal-5'-phosphate (P-5-P) all inhibited the ATPase activity in a concentration-dependent manner with a potency order of ARL 67156 = PPADS>P-5-P. In conclusion, EFS of several tissues in which ATP is a neurotransmitter causes the release of an ATPase and activity is greatest in the guinea-pig vas deferens. The enzyme has pharmacological and kinetic characteristics that are similar to ectonucleoside triphosphate diphosphohydrolases.