We have examined the interaction between synapsin I, the major phosphoprotein on the membrane of small synaptic vesicles, and brain spectrin. Using recombinant peptides we have localized the synapsin I attachment site upon the beta-spectrin isoform betaSpIISigmaI to a region of 25 amino acids, residues 211 through 235. This segment is adjacent to the actin binding domain and is within the region of the betaSpIISigmaI that we previously predicted as a candidate synapsin I binding domain based upon sequence homology. We used differential centrifugation techniques to quantitatively assess the interaction of spectrin with synaptic vesicles. Using this assay, high affinity saturable binding of recombinant betaSpIISigmaI proteins was observed with synaptic vesicles. Binding was only observed when the 25 amino acid synapsin I binding site was included on the recombinant peptides. Further, we demonstrate that antibodies directed against 15 amino acids of the synapsin I binding domain specifically blocked synaptic transmission in cultured hippocampal neurons. Thus, the synapsin I attachment site on betaSpIISigmaI spectrin comprises a approximately 25 amino acid segment of the molecule and interaction of these two proteins is an essential step for the process of neurotransmission.