T51B rat liver cells and several carcinogen-treated, carcinogen + saccharin-treated, or spontaneously altered clones of T51B cells were tested for their abilities to form colonies in calcium-deficient medium and soft agar and to produce tumors in athymic nude mice. Most (10 out of 11) of the clones which were derived from colonies in calcium-deficient medium were unable to form colonies in soft agar and 8 out of 11 were non-tumorigenic. Conversely, 6 out of 9 clones derived from colonies in soft agar were unable to multiply significantly in calcium-deficient medium and 5 of these 6 clones were also non-tumorigenic. Two of these 9 soft agar-growing clones were tumorigenic, one of which also proliferated in calcium-deficient medium, and the other of which acquired the ability to proliferate in calcium-deficient medium after it became able to form tumors in athymic nude mice. Thus, T51B rat liver cells gain the ability to grow in calcium-deficient medium and soft agar independently during the process of neoplastic transformation and neither characteristic by itself reliably predicts tumorigenicity.