Effects of 1, 25-dihydroxyvitamin D₃ (1, 25(OH)₂D₃), recombinant human transforming growth factor (rhTGF)-β₁ and recombinant human bone morphogenetic protein (rhBMP)-2 on differentiation in four different canine osteosarcoma cell lines (POS53B, 53C, 53D and 14A) were examined using markers specifically expressed by phenotypic osteoblasts. 1, 25(OH)₂D₃ increased alkaline phosphatase (ALP) activity in one cell line, osteocalcin production in two lines and type I collagen production in three lines. RhTGF-β₁ increased ALP activity in one clonal cell, osteocalcin production in one clonal cell and type I collagen production in two clonal cells. RhBMP-2 increased ALP activity in all clonal cells, osteocalcin production in two clonal cells and type I collagen production in three clonal cells. Thus, these agents induced differentiation in osteosarcoma cells at different efficacies. Electron microscopic study revealed that these agents increased cellular activity in all cell lines with no evidence of degeneration of cell organelle by drug cytotoxicity. In some cultures treated with either 1, 25(OH)₂D₃ or rhBMP-2, apoptotic cells were observed. Based on the change in markers, rhBMP-2 and 1, 25(OH)₂D₃ seemed to be more effective than rhTGF-β₁. These agents are potential inducers of apoptosis.