Immunohistochemical and histochemical stains are useful adjunct techniques in the diagnosis of canine cutaneous round cell tumors, which can appear histologically similar. We applied a panel of monoclonal antibodies (recognizing tryptase, chymase, serotonin for mast cells; CD1a, CD18, MHC class II for histiocytes; CD3 for T lymphocytes; CD79a for B lymphocytes and plasma cells) and one histochemical stain (naphthol AS-D chloroacetate for chymase activity) to formalin-fixed, paraffin-embedded sections of canine cutaneous mast cell tumors, histiocytomas, lymphosarcomas, plasmacytomas, and unidentified round cell tumors. Of 21 tumors with a histologic diagnosis of mast cell tumor, 7/7 (100%) grade I, 6/7 (85.7%) grade II, and 3/7 (42.9%) grade III tumors were diagnosed as mast cell tumors based on positive staining for tryptase antigen and chymase activity. Mast cells were positive for both tryptase antigen and chymase activity, indicating equal efficacy of tryptase immunohistochemistry and chymase histochemistry. Chymase was detected immunohistochemically in both tumor and nontumor cells, while serotonin was not detected in most mast cell tumors, and thus, neither was useful in the diagnosis of mast cell tumors. Immunohistochemistry to detect CD18 and MHC class II was equally effective in staining histiocytomas, although lymphosarcoma must be ruled out through the use of CD3 and CD79a immunohistochemistry. Immunohistochemistry using three different monoclonal antibodies to human CD1a showed no cross-reactivity in canine histiocytomas and was not useful. A final diagnosis was obtained for 4/5 (80%) of the unidentified tumors, indicating the usefulness of multiple stains in poorly differentiated round cell tumors.