INT6 is a unique gene, highly conserved throughout evolution and associated with mammary tumorigenesis in the mouse. Although it is expressed in all adult tissues of the mouse and early in embryonic development, its function is unknown. To study the normal distribution and the potential function of the Int6 gene products, we produced antibodies against synthetic peptides specific for the Int6 protein. Western blot and immunoprecipitation analysis demonstrated a 43-kD major gene product that is localized in the cytosolic fraction of mammary cell homogenates. This latter observation is supported by immunoperoxidase analysis, which shows a strong staining anti-Int6 peptide in the perinuclear region of the HC11 mammary epithelial cell line, suggesting a possible localization in the Golgi apparatus. Further immunocytochemical studies in the mouse embryo show that Int6 expression is prevalent in migrating neural crest cells, in the notochord, and in condensing cartilage between 9.5 and 14.5 days of development. In these embryonic tissues, Int6 staining co-localizes with the staining of ricinus lectin, and giantin, proteins that are specifically associated with the Golgi apparatus. The restricted expression of the protein within the Golgi apparatus and its strong conservation throughout evolution suggest that Int6 may perform an essential cellular function.