Cholinergic stimulation of parietal cell secretion is mediated by an increase in intracellular calcium. KN-62, a selective inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK II), has recently been synthesized (Tokomitsu et al. J. Biol. Chem. 265: 4315-4320, 1990). To define the role of CaMK II in parietal cell secretion, we determined the effects of KN-62 on secretagogue-stimulated acid secretion in isolated rabbit parietal cells. Pretreatment of parietal cells with KN-62 resulted in the inhibition of carbachol-stimulated [14C]aminopyrine uptake over a concentration range of 3 to 60 microM (IC50 of 20 microM). KN-62 (60 microM) reduced carbachol-stimulated aminopyrine uptake to unstimulated levels. KN-62 did not alter carbachol-stimulated increases in cytoplasmic free Ca2+ concentration. High concentrations of KN-62 (60 microM) elicited a small decrease in aminopyrine uptake stimulated by forskolin, but did not significantly inhibit histamine stimulation. A potent CaMK II activity was identified in total membrane from parietal cells. These results suggest that CaMK II may mediate cholinergic-stimulated parietal cell secretion.