The synaptotagmins are integral membrane proteins of synaptic vesicles thought to serve as Ca2+ sensors in the process of vesicular trafficking and exocytosis. Results from antibody microinjection and gene-disruption experiments have led to a controversy over whether synaptotagmins are essential for neurotransmission. However, the studies casting doubt on the role of synaptotagmins have assumed that no further isoforms of these molecules exist. Here we report the isolation of a third member of the synaptotagmin family (Syt3) from mouse brain. Although retaining the characteristic five-domain structure of the other synaptotagmins, SYT3 is considerably more divergent at the level of amino acid sequence. In the most highly conserved C2 domain, the mammalian synaptotagmins, SYT1 and SYT2, share 88% sequence identity, whereas SYT3 has only approximately 45% identity to either. Overall, SYT3 has the greatest sequence identity with rat SYT2 and marine ray p65A (both 37%), although homology to all of the known synaptotagmins is > 30%. However, SYT3 is most like p65C when comparing domain structure. Syt3 is expressed in many regions of the nervous system but is undetectable in extraneural tissues. The three murine synaptotagmins have differential expression patterns in the brain. Furthermore, in PC12 cells, Syt3 is coexpressed with Syt1 and is more abundant than the latter. This result suggests that individual neurons may have specific combinations of synaptotagmins that could provide for diversity in vesicular release.