Calmodulin is an important element in the regulation of nerve terminal exocytosis by Ca[sup2+]. Calmodulin has been shown to interact with the synaptic vesicle phosphoproteins synapsins Ia and Ib (Okabe, T. & Sobue, K. (1987) FEBS Lett. 213, 184-188; Hayes, N. V. L., Bennett, A. F. & Baines, A. J. (1991) Biochem. J. 275, 93-97). These proteins are thought to provide regulated linkages between synaptic vesicles and cytoskeletal elements. It is well established that calmodulin modulates synapsin I activities via calmodulin-dependent protein-kinase-II-catalysed phosphorylation. The direct binding of calmodulin to synapsin I suggests a second mode of regulation in addition to phosphorylation. In this study, we present evidence indicating that two sites for calmodulin binding exist in the N-terminal head region of synapsins Ia and Ib. In unphosphorylated synapsin I, these sites had a K[subd] value of = 36 ± 14 nM for binding to calmodulin labeled with acetyl-N'-(5-sulpho-1-naphthyl)ethylene diamine. The K[subd] values for synapsin I phosphorylated at various sites were as follows: site I 18 ± 11 nM; sites II and III 35 ± 14 nM; sites I-III 16 ± 9 nM. The fluorescence data indicated a stoichiometry of not less than 2 mol calmodulin bound to 1 mol synapsin I at saturation in each case. Consistent with this stoichiometry, two chemically cross-linked species (96 Kda and 116 Kda) containing calmodulin and synapsin I were generated in vitro, corresponding to one and two calmodulin molecules bound/synapsin I. Defined fragments of synapsin I were generated with the reagent 2-nitro-5-thiocyanobenzoic acid, which cleaves at cysteine residues. Cysteine-specific cleavage of whole synapsin I after cross-linking to biotinylated calmodulin generated a pair of polypeptide complexes (approximately 46 Kda and 38 Kda), the masses of which indicated cross-linking of calmodulin to the N-terminal and middle regions of synapsin I. [ABSTRACT FROM AUTHOR]