Three calcium channel antagonists, verapamil, diltiazem and nisoldipine, inhibited superoxide production in human neutrophils that were stimulated by phorbol 12-myristate 13-acetate (PMA) in a buffered saline lacking calcium. Concentrations of these drugs giving 50% control activity (IC50) were 0.3, 0.45 and 0.01 mM respectively. This inhibition was also observed in the presence of ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) and was not reversed by the addition of calcium. This suggests that calcium channel antagonists inhibited superoxide production independently of extracellular calcium. These calcium channel antagonists inhibited the mobilization of membrane-associated calcium, and protein phosphorylation probably catalyzed by C-kinase, both of which are thought to be involved in the signal transmission for the induction of superoxide production. Calcium channel antagonists also inhibited NADPH oxidase, responsible for superoxide production, with IC50 = 0.5, 3 and more than 0.08 mM, respectively, for verapamil, diltiazem and nisoldipine. The results indicate that calcium channel antagonists inhibit superoxide production by affecting not only the catalytic activity by also the activation of NADPH oxidase. Inhibition of superoxide production by calcium channel antagonists suggests that these antagonists do not affect cell functions merely by affecting calcium influx.