The calmodulin-binding polypeptide neuromodulin (GAP-43) was tested in vitro for its ability to modulate a typical calmodulin target, the enzyme nitric oxide synthase. The titration of enzyme with increasing neuromodulin concentrations demonstrated a concentration-dependent decrease in enzyme activity. Subsequent analysis of the ability of increased calcium concentrations to activate the enzyme was tested in the presence or absence of neuromodulin. The effect of neuromodulin on the calcium-dependent activation of the enzyme was to depress enzyme activity in the range of 0.2 to approximately 6 microM calcium. Treatment of the neuromodulin polypeptide with protein kinase C eliminated its ability to inhibit nitric oxide synthase activation. Subsequent treatment of the phosphorylated neuromodulin with calcineurin (phosphatase 2b) caused it to regain its inhibitory action on the enzyme. The results from these in vitro studies have indicated that neuromodulin has the ability to affect the activation of a calmodulin-dependent enzyme at levels of the polypeptide that exist in neurons. They also demonstrated that the regulation occurred within a physiological range of calcium concentrations. Since the inhibition of enzyme activity appeared to be occurring through the interaction of neuromodulin with calmodulin, it seems likely that neuromodulin has a general ability to impede activation of calmodulin-dependent targets.