This study aimed to detect in vivo expression of human melanoma-associated antigen D-1, which was identified by screening an expression cDNA library constructed from mRNA extracted from cultured melanoma cells with sera from patients with melanoma. The tissue distribution of D-1 antigen was then analyzed. Murine anti-D-1 recombinant peptide polyclonal antibodies were raised by immunization of in vitro synthesized D-1 peptide against Balb/c mice and applied immunohistochemically on paraffin-embedded tissue specimens. D-1 antigen was found to be restrictedly expressed on melanoma cells, but not on normal melanocytes, adjacent keratinocytes, fibroblasts, lymphocytes and adnexal structures of skin. The reactivities of anti-D-1 antibodies did not correlate with histogenesis of the lesions, their ability to produce melanin, and/or their primary or metastatic nature. There was no positive reactivity of anti-D-1 antibodies with other skin tumors, including squamous cell carcinoma, basal cell epithelioma, seborrheic keratosis, and nevus cell nevus. Further, cytoplasmic expression of D-1 antigen in melanoma cells was observed only in a certain subgroup of patients with melanoma. This indicates that the cell surface expression of D-1 peptide requires specific transporting proteins, such as HLA molecules.