The SNARE hypothesis of vesicular traffic proposes that three proteins, VAMP/synaptobrevin, syntaxin, and SNAP-25, constitute a complex that docks the vesicle at the target membrane. VAMP and syntaxin isoforms have been identified outside the nervous system, and a cDNA to a SNAP-25 related protein, SNAP-23, was recently identified in human lymphocytes. Here we report the generation of isoform-specific antibodies to SNAP-23 cloned from human melanoma cells, and their use in detecting the expression and localization of the endogenous SNAP-23 protein in several tissues and cell lines. SNAP-23 was readily detected in liver, lung, kidney, and spleen, to a lesser extent in muscle and heart, and was almost undetectable in brain. The protein was also abundant in fibroblast, muscle, and fat cell lines, but relatively less enriched in neuroendocrine PC12 cells. SNAP-23 abundance did not change during differentiation of 3T3-L1 fibroblasts into adipocytes. In both, SNAP-23 was membrane-bound and below detectable levels in the cytosolic fraction. Subcellular fractionation of 3T3-L1 adipocytes revealed that the majority of the protein was associated with plasma membranes. These findings support the conclusion that a tripartite SNARE complex exists outside of the nervous system, and suggest that SNAP-23 may play a role in vesicle traffic in most cell types.