Electrolyte fluxes are fundamental to normal endocrine pancreatic function. Adenosine triphosphatases (ATPases) are enzyme systems believed to modulate electrolyte movements across membranes in a number of cell types. This study was undertaken to measure cation-dependent ATPases of rat pancreatic islets. In addition, we compared effects of substances which influence endocrine pancreatic function upon ATPases in homogenates of islets and kidney, the latter being a tissue which would not be expected to have a stimulus-secretion response to substances which activate islets. Both tissues were generally similar with respect to apparent Michaelis constant (ATP) of Na(+)K(+)ATPase, Mg(++)ATPase, and Ca(++)ATPase. In islets and kidney, Na(+)K(+)ATPase specific activity was increased when the Na:K ratio was lowered from 250:1 (175:0.7 mM) to 5:1 (100:20 mM). Inhibition of Na(+)K(+)ATPase at either Na:K ratio by ouabain, an activator of secretion, and enhancement of the high-ratio Na(+)K(+)ATPase by diphenylhydantoin, an islet secretory inhibitor, were also common to both tissues. Because both inhibition and enhancement of Na(+)K(+)ATPase could be studied at the high Na:K ratio, we examined the effect of regulators of secretion upon the activity of this enzyme. Like ouabain, substances which induce or support islet secretion, glucose 16 mM or 3.3 mM, arginine 14.2 mM (with 3.3 mM glucose), or Ca(++) 1 mM, inhibited high-ratio islet Na(+)K(+)ATPase. Like diphenylhydantoin, the inhibitors of insulin secretion, diazoxide 0.22 mM, or NH(4)Cl 16 mM, enhanced this islet ATPase. Neither valine, which is non-secretogenic, nor arginine without glucose, which is a weak secretagogue, had any effect upon islet Na(+)K(+)ATPase. We examined the effect of these substances upon other cation-dependent islet ATPases. Ca(++) inhibited Mg(++)ATPase, and glucose inhibited Ca(++)ATPase. Leucine, 22.9 mM, which induces insulin secretion in the absence of glucose, suppressed islet Ca(++)ATPase and had no effect upon high-ratio Na(+)K(+)ATPase. In contrast to the observations in the islets, most substances which influence islet function had no effect on kidney ATPases, or effects which were different from those seen in islets. Except for ouabain, none of these substances influenced the three kidney ATPases in a manner similar to that seen with islets. These findings support the hypothesis that cation-dependent ATPases are involved in specificity of islet response to substances which influence endocrine pancreatic activity.