The cellular mechanism that underlies the regulated release of synaptic vesicles during neurotransmission is not fully known. Our previous data has shown that brain spectrin (alphaSpIIsigma1/betaSpIIsigma1)2 is localized in axons and nerve terminals and we have shown that the beta subunit (betaSpIIsigma1) contains a synapsin-binding domain capable of interacting with synapsin and small synaptic vesicles in vitro and in vivo. These findings suggested a role for brain beta-spectrin in synaptic neurotransmission. To examine this possibility further, peptide-specific antibodies directed against epitopes within the synapsin-binding domain of brain beta-spectrin, or against flanking regions, were injected into the presynaptic neuron of synaptically paired rat hippocampal neurons in culture. Here, we show that the antibodies directed against the synapsin-binding domain specifically blocked synaptic neurotransmission.