PNL2 is a recently generated monoclonal antibody (mAb) that recognizes normal and neoplastic melanocytes. Although the antigen recognized by PNL2 remains unknown, recent studies of human and mouse melanomas have confirmed its usefulness as a diagnostic marker. In the current study, the immunoreactivity of PNL2 in canine melanomas was tested and compared with Melan A (A103). Validation of PNL2 was performed by Western blot analysis. PNL2 and Melan A immunoreactivity were tested on frozen samples of canine melanomas and on 69 formalin-fixed, paraffin-embedded melanocytic neoplasms. Normal canine tissues and nonmelanocytic neoplasms were included as negative controls. Western blot confirmed the presence of a protein recognized by the PNL2 antibody in canine melanomas. Immunohistochemically, PNL2 stained the melanocytic neoplastic cells with an intracytoplasmic, granular pattern. Among the melanocytic neoplasms tested, 62% stained positively with PNL2 and 59% with Melan A; 50.7% stained positively with both mAbs. The overall percentage of neoplasms that stained positively with at least 1 of these 2 antibodies was 68%. The extent of staining (i.e., the percentage of cells stained per specimen) was greater with PNL2 than with Melan A. With both mAbs, staining was most intense and diffuse in the epithelioid cell phenotype. Neither nonspecific staining nor staining in cells other than melanocytes was detected with either mAb. In contrast to human granulocytes, canine granulocytes were negative by both Western blot and immunohistochemical analyses. PNL2 mAb proved to be highly specific for the identification of formalin-fixed canine melanocytic neoplasms and should be a valuable diagnostic reagent.