A transformation assay system using two clonal strains of adult rat liver epithelial cells is described. The carcinogens N-methyl-N'-nitro-N-nitrosoguanidine, methylmethanesulfonate, N-2-fluorenylacetamide, aflatoxin B1, benzo(a)pyrene, dimethylnitrosamine, nitrosopyrrolidine, and dimethyl-4-aminoazobenzene induced anchorage independency in one or both clonal strains, whereas noncarcinogenic analogues were inactive. In the absence of carcinogen exposure, the cell strains exhibited no spontaneous transformation to anchorage independency. These results demonstrate the reliability of anchorage independency as an in vitro end point and the specific responsiveness of adult rat liver epithelial cells to a wide range of carcinogens. Thus, the assay of transformation in adult rat liver epithelial cells is potentially useful for detection of chemical carcinogens and the study of their mechanisms of action.