Using a new subtraction method and chemically induced rat hepatocellular carcinomas, we identified a hepatocellular carcinogenesis and hepatocyte proliferation-related gene designated hp33 that encoded a 33-kDa protein. The predicted protein was similar to the bovine aralkyl N-acyltransferase and arylacetyl N-acyltransferase. HP33 was restrictively expressed in the liver and kidney, and its gene expression was stimulated in the regenerating liver as well as in hepatocellular carcinoma. Interestingly, it was demonstrated in various hepatic cells that HP33 was localized in regions surrounding the centrosome, where mitochondria were not concentrated. Moreover, its centrosomal localization was evident in the interphase but not in the mitotic phase of the cell cycle. The centrosomal localization of HP33 was dependent on microtubules, and ectopically expressed HP33 was seen at centrosomes even in fibroblasts, which do not exhibit a typical staining pattern of HP33. The centrosomal localization of HP33 became invisible by nocodazole treatment, whereas the mitochondrial staining pattern was not affected by it. In vitro cosedimentation experiments using purified microtubules indicated that HP33 bound to MTs directly and that its MT-binding ability was dependent on the C-terminal basic domain of the protein. These results suggest that, different from early predictions based on its primary structure, HP33 has a growth- and carcinogenesis-related function that may be independent of mitochondrial function.