Seven monoclonal antibodies (Moabs), recognizing melanoma-associated antigens in human tissues, were evaluated for their ability to immunohistochemically stain formalin-fixed, paraffin-embedded canine melanomas. Only 2 Moabs, designated human melanosome-specific antigen (HMSA)-1 and HMSA-5, stained routinely processed canine melanomas, staining 21/35 (60%) and 24/35 (69%), respectively. Twenty-nine of 35 (83%) melanomas tested were stained if results of the 2 Moabs were combined. Monoclonal antibody HMSA-1 also stained neoplastic cells of 10/35 (29%) tumors of nonmelanocytic origin and some neurons and salivary gland epithelial cells in normal canine tissues. However, Moab HMSA-1 staining in the nonmelanocytic tumors, consisting of small, discrete periodic acid-Schiff-positive cytoplasmic droplets, was readily distinguishable from the diffusely granular, cytoplasmic staining of melanocytic tumors. In addition to melanomas, Moab HMSA-5 stained melanocytes and some melanin-containing tumor cells of a pigmented basal cell tumor and melanocytes in normal canine skin. Monoclonal antibodies HMSA-1 and HMSA-5 immunohistochemically identified the majority of canine melanomas, had limited and distinguishable staining in normal tissues and nonmelanocytic tumors, and therefore may be a useful adjunct for the diagnosis of canine melanoma in formalin-fixed, paraffin-embedded tissues.