A cell line (HRRT) derived from a hereditary renal rat tumor has been used in an assay for initiators and promoters of carcinogenesis based on attachment-independent survival in aggregates. Treatment with single noncytotoxic doses of the carcinogens urethan (1 mM), N-methyl-N-nitrosourea (30 microM), and benzo(a)pyrene (0.2 microM) for 1 hr did not affect survival of HRRT cells in the aggregate assay system. However, when carcinogen treatment was followed by exposure of the cells to the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (0.16 microM), a considerable increase in survival was observed. With urethan as an initiator, it was found that tumor promoters (12-O-tetradecanoylphorbol-13-acetate and phorbol-12, 13-didecanoate) induced a considerable response in the assay system, while nonpromoting phorbol esters (4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and 4 alpha-phorbol-12, 13-didecanoate) did not affect the survival. Exposure of HRRT cells to NiSO4 (40 microM) for 3 hr did not influence cell survival in the aggregate form. However, subsequent treatment of the cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate induced a marked increase in the number of viable cells. Moreover, treatment of HRRT cells with a nontransforming dose of urethan (1 mM) for 1 hr followed by continuous exposure to nickel sulfate (40 microM) also increased cell survival in the aggregate form. These results support the view that nickel sulfate may act as both an initiator and a promoter in mammalian cell transformation. The present data also indicate that the aggregation assay system using the HRRT cell line may be a valuable in vitro screening assay for putative initiators and promoters of carcinogenesis.