Munc13-1, a mammalian homologue of Caenorhabditis elegans unc-13p, is a presynaptic phorbol ester receptor that enhances neurotransmitter release. In the present study we analysed the regional, cellular and subcellular expression patterns in rat of two novel Munc13 proteins, Munc13-2 and Munc13-3. We demonstrate by hybridization in situ that Munc13-1 mRNA is expressed throughout the brain, whereas Munc13-2 mRNA is preferentially present in rostral brain regions, and Munc13-3 mRNA in caudal areas. In an analysis of subcellular brain fractions with isoform-specific antibodies, we show that the novel Munc13 proteins are enriched in synapses. Immunocytochemical examination of rat cerebellar sections indicates that Munc13-3, like Munc13-1, is concentrated in presynaptic terminals. Our results characterize Munc13 proteins as a family of neuron-specific, synaptic molecules that bind to syntaxin, an essential mediator of neurotransmitter release. Munc13-2 and Munc13-3 are expressed in a complementary fashion and might act in concert with Munc13-1 to modulate neurotransmitter release.