Nucleoside diphosphate kinase (NDPK) participates in multiple cellular functions, yet the molecular mechanisms of its involvement are often unknown, given that there are no specific inhibitors for the enzyme from vertebrates. We developed antibodies against NDPK by immunization of rabbits with the enzyme from bullfrog skeletal muscle. The antibodies specifically recognized the enzyme from frog tissues, and cross-reacted with NDPK from Xenopus. In contrast to mammalian NDPK, the amphibian enzyme elicited antibodies that inhibit potently its catalytic function. We utilized the inhibitory properties of these immunoglobulins to examine the role of NDPK on the ATPgammaS-induced stimulation of Ca2+ and K+ currents of cardiac myocytes. Injection of NDPK-neutralizing Fab fragments into atrial cells reduced considerably the effect of ATPgammaS on muscarinic K+ currents, but not on Ca2+ currents. Therefore, ATPgammaS increases calcium and potassium currents of atrial cells by two distinct mechanisms. NDPK is essential for the conversion of ATPgammaS into GTPgammaS which leads to muscarinic K+ channel activation but not for the stimulation of Ca2+ currents by ATPgammaS. The results demonstrate that antibodies to frog NDPK block the activity of the enzyme in vivo and in vitro, and can be used to determine the relevance of NDPK and its catalytic activity to the function of vertebrate cells.