The addition of Ca2+, adenosine 5'-[gamma-thio]triphosphate (ATP[S]) or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) to digitonin-permeabilized PC12 cells stimulates noradrenaline secretion. Both ATP[S] and GTP[S] stimulate release in the absence of Ca2+. Whereas ADP and adenosine 5'-[beta gamma-imido]triphosphate inhibited ATP[S]-stimulated release, they did not inhibit Ca(2+)-stimulated release even in the absence of added ATP. This suggests that the kinase which uses ATP[S] to induce secretion may not play an essential role in Ca(2+)-stimulated release. As GTP[S]-stimulated and ATP[S]-stimulated secretions were not additive, it seemed possible that stimulation by ATP[S] might in part result from the thiophosphorylation of GDP by nucleoside-diphosphate (NDP) kinase to form GTP[S]. The following results are consistent with this possibility. (1) A low concentration of GDP increased ATP[S]-stimulated secretion, but not GTP[S]-stimulated or Ca(2+)-stimulated secretion. (2) A variety of ribo- and deoxyribo-nucleoside di- and tri-phosphates inhibited ATP[S]-stimulated secretion, but not GTP[S]-stimulated or Ca(2+)-stimulated secretion. Thus, like NDP kinase, the kinase which uses ATP[S] to cause noradrenaline release appears to have a very low specificity for ATP. (3) Incubation of permeabilized cells in a sucrose-containing buffer resulted in the preferential loss of ATP[S]-stimulated secretion and a decrease in the level of NDP kinase. The addition of rat liver NDP kinase to those depleted cells partially restored ATP[S]-stimulated secretion.