Six clonal cells were established from a canine osteosarcoma cell line (POS) by a limiting dilution method using feeder cells. Whereas histology of mass developed in nude mice by transplantation of POS cells revealed various cell types, clonal cells were morphologically consistent in size and shape. Doubling time of clonal cells ranged from 30 +/- 1.4 to 54 +/- 1.3 hr and alkaline phosphatase activity ranged from 0.040 +/- 0.001 to 2.61 +/- 0.435 mumol/min/mg protein depending on the cell types. When transplanted into nude mice, each clonal cell type formed following four histological types; osteoblastic, chondroblastic, fibroblastic, and undifferentiated types. Since each histological feature was found simultaneously in the primary tumor, osteosarcoma tissue might be a complex of various types of cells having different characteristics. Therefore, POS clonal cells may be useful as a potential tool for the studies of differentiation, phenotypic expression, and a new therapeutic modality of osteosarcoma.