Syntaxin 17 (STX17) is a divergent member of the syntaxin family of proteins first discovered by Scheller and colleagues in a yeast two-hybrid screen designed to identify novel mammalian SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors). We recently independently identified STX17 as a novel Ras-interacting protein, but immunohistochemical studies suggested that STX17 is localized to the nucleus in normal pancreatic ductal epithelial, acinar, and islet cells in contrast to previous reports of cytoplasmic localization, albeit in other cell types. Therefore, we have conducted a more thorough survey of various human and mouse tissues to better establish the expression pattern of STX17 in different tissues and cell types. Although RT-PCR experiments demonstrate ubiquitous expression of STX17, closer examination by immunohistochemistry reveal that STX17 expression is limited to certain cell types. Furthermore, in contrast to the cytoplasmic localization previously reported in a limited number of cell types, we find that in many other cell types, syntaxin 17 can be found in the nucleus. Finally, we demonstrate that in human hepatocellular carcinoma cell lines, STX17 localization is altered relative to normal hepatocytes, although the localization of STX17 differs even between these established human cancer cell lines and fresh human hepatocellular carcinoma cells, emphasizing the caution that must be exercised in drawing conclusions from data gathered in cell lines. The sequence divergence of STX17, the unexpected nuclear localization of STX17 in many cell types, and the altered localization of STX17 in malignant cells argue for a novel function of syntaxin 17 distinct from its hypothesized role in mediating membrane fusion events.