In this study, we examined the effects of four chemical reagents, 5 azacytidine (5azaC), all-trans-retinoic acid (ATRA), Phorbol 12-myristate 13-acetate (TPA) and trichostatin A (TSA), on an MCM-B2 canine mammary tumor cell line. Growth of the MCM-B2 cells was inhibited by addition of any of these four chemical reagents in a dose-dependent manner. TPA-treated cells became blast-cell-like and had high expressions of cyclin D, cyclin B and PCNA. Both ATRA-treated and 5azaC-treated cells showed decreased expression of cell cycle related molecules and increased expressions of the mammary epithelial marker cytokeratin 18 and underwent morphological changes. ATRA-treated cells were converted from the originally spindle-shaped cells into cubic-shaped cells, and 5azaC-treated cells became fibroblast-like. In the TSA-treated cells, the cytoplasm was enlarged, increasing the cytoplasm/nucleus ratio, and the expressions of cell cycle-promoting molecules (cyclin D and PCNA), as well as cell cycle-inhibiting factors (p21(WAF1) and p27(kip1)), were increased. These results demonstrated the growth-inhibiting, differentiation-inducing and antitumor effects of each of these four chemicals on the unique phenotype of canine mammary tumors.