Tumorigenic cell lines were established in culture from three transplantable mouse colonic carcinomas designated CT 26, CT36, and CT 51. The cultured lines were characterized for the retention of the biological characteristics of the parental lines. All three cultured lines retained the ability to form tumors in vivo. Serially transplanted parental lines CT 26 and CT 51 grew at a faster rate than did CT 36 and showed a greater propensity for the formation of lung metastases. Similar characteristics were exhibited by the tumors formed from the injection of cultured cells. The cultured cell lines were also evaluated with respect to a number of in vitro markers for cancer. Cultured CT 26 and CT 51 cells formed tumors at lower inocula than did CT 36. CT 26 and CT 51 showed anchorage-independent growth and lack of contact inhibition, while CT 36 grew as a strict monolayer and did not form colonies in 0.27% agarose. CT 26 had the highest saturation density of the cell lines when grown in media supplemented with either 10 or 2.5% fetal bovine serum, while CT 51 had the lowest saturation density under these conditions. The varying degrees of malignancy exhibited by the three cell lines and the overall retention of the biological characteristics of the parental lines by the cultured lines suggest that the cultured cells (without the contaminating stromal elements present in the serially transplanted lines) will provide suitable material for the investigation of the molecular bases of these malignant characteristics.