Microvesicles are endocrine counterparts of neuronal synaptic vesicles, and accumulate and secrete classic neurotransmitters. In mammalian pinealocytes, microvesicles accumulate L-glutamate through a vesicular glutamate transporter and secrete it through exocytosis. To characterize the molecular organization of microvesicles in more detail, we investigated in this study the expression and localization of synaptic vesicle protein 2 (SV2) in rat pinealocytes. RT-PCR analysis indicated that transcripts specific for two isoforms, SV2A, a ubiquitous form present in neuronal and endocrine cells, and SV2B, a neuron-specific form, are amplified in pineal RNAs. Northern blotting with specific transcripts indicated that the mRNA for SV2B is predominantly expressed, whereas that for SV2A is below the detection limit. Site-specific antibodies against SV2B recognized a single 72-kDa polypeptide in the pineal membrane fraction, whereas anti-SV2A antibodies did not recognize any polypeptides. Immunohistochemical analysis of cultured cells indicated that SV2B is expressed in pinealocytes but not in other types of cells. SV2B is present in somata and is especially rich in processes, which are filled with microvesicles. SV2B is colocalized with synaptophysin and synaptotagmin, markers for microvesicles. Immunoelectron microscopy indicated that SV2B is associated with microvesicles. These results indicated that SV2B, but not SV2A, is expressed in rat pinealocytes and associated with microvesicles. As SV2B is also expressed in cultured alphaTC6 clonal pancreatic a cells, SV2B is not a protein specific for neurons.