Overexpression of frequenin and its orthologue neuronal Ca(2+) sensor 1 (NCS-1) has been shown to increase evoked exocytosis in neurons and neuroendocrine cells. The site of action of NCS-1 and its biochemical targets that affect exocytosis are unknown. To allow further investigation of NCS-1 function, we have demonstrated that NCS-1 is a substrate for N-myristoyltransferase and generated recombinant myristoylated NCS-1. The bacterially expressed NCS-1 shows Ca(2+)-induced conformational changes. The possibility that NCS-1 directly interacts with the exocytotic machinery to enhance exocytosis was tested using digitonin-permeabilized chromaffin cells. Exogenous NCS-1 was retained in permeabilized cells but had no effect on Ca(2+)-dependent release of catecholamine. In addition, exogenous NCS-1 did not regulate cyclic nucleotide levels in this system. These data suggest that the effects of NCS-1 seen in intact cells are likely to be due to an action on the early steps of stimulus-secretion coupling or on Ca(2+) homeostasis. Myristoylated NCS-1 bound to membranes in the absence of Ca(2+) and endogenous NCS-1 was tightly membrane-associated. Using biotinylated NCS-1, a series of specific binding proteins were detected in cytosol, chromaffin granule membrane, and microsome fractions of adrenal medulla. These included proteins distinct from those detected by biotinylated calmodulin, demonstrating the presence of multiple specific Ca(2+)-independent and Ca(2+)-dependent binding proteins as putative targets for NCS-1 action. A model for NCS-1 function, from these data, indicates a constitutive membrane association independent of Ca(2+). This differs from the Ca(2+) myristoyl switch model for the closely related recoverin and suggests a possible action in rapid Ca(2+) signal transduction in response to local Ca(2+) signals.