We have previously reported that GDP-bound alpha beta gamma-trimeric GTP-binding (G) proteins can be converted into the active GTP-bound form with nucleoside diphosphate (NDP) kinase and ATP, although its exact activation mechanism still remains to be resolved. In the present study, we investigated whether NDP kinase activity was modified by mastoparan, a wasp venom peptide that is known to activate G proteins as an agonist-receptor complex. The activity of NDP kinase measured by the formation of GTP from ATP and GDP was markedly stimulated, when the kinase was incubated with mastoparan. The concentration of mastoparan required for the activation was much lower than that observed for the peptide-induced activation of G proteins under similar assay conditions. There was also an increase in the phosphorylated intermediate of NDP kinase as well as the catalytic activity upon its incubation with mastoparan. These results suggest that mastoparan not only activates G proteins directly via guanine nucleotide exchange reaction but also stimulates NDP kinase activity.