Synaptotagmins are a large family of membrane-trafficking proteins. They are evolutionarily conserved and have 15 members in rodents and humans. Synaptotagmins-1, -2, and -9, are known to have an essential role as calcium sensors for fast synaptic release. Synaptotagmin-7 is a major calcium sensor for the exocytosis of large secretory vesicles in endocrine cells. The functional roles of most synaptotagmin isoforms remain unknown. Here we examined whether synaptotagmins are expressed in the rodent brain in distinct patterns and whether individual neurons and astrocytes coexpress multiple synaptotagmin isoforms. We performed a systematic analysis of expression using radioactive in situ hybridization and quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) as well as multiplex RT-PCR on a single-cell level. Our results demonstrate that most synaptotagmins are expressed in the rodent brain in highly distinctive expression patterns, and that individual neurons express variable subsets of different synaptotagmins. We also show that Syt-11 is the major isoform expressed in astrocytes. This study therefore supports the hypothesis that the functional properties of individual neurons and astrocytes are conferred by the specific subset of synaptotagmins expressed in a cell.