Recombinant human MART-1 protein was produced by bacterial and baculoviral-insect cell expression systems. By immunization with bacterial MBP-MART-1 fusion protein or MBP cleaved MART-1 protein, a rabbit polyclonal and two murine monoclonal antibodies specific for MART-1 were produced. These antibodies specifically detected MART-1 in immuno-precipitation, Western blotting, flow cytometric assays and in immunohistochemical analysis of tissue sections. They also stained cytoplasmic components in melanocytes and most melanoma cells in frozen or paraffin embedded tissue sections, indicating that these antibodies may be useful for the diagnosis of melanoma. One of the monoclonal antibodies M2-7 C10 recognized only human MART-1, but the other monoclonal antibody M2-9 E3 recognized both human and murine MART-1. The size of the human MART-1 molecule detected by SDS-PAGE with these antibodies was approximately 18 kDa, suggesting possible posttranslational modifications in the MART-1 protein. Subcellular fractionation studies suggested that MART-1 was present in melanosomes and endoplasmic reticulum, although known melanogenic enzymatic activities were not detected in the MART-1 protein. These reagents may be useful for biological studies on melanocytes and melanoma cells as well as for the development and monitoring of immunotherapy for patients with melanoma.