The purpose of the studies reported here was to compare the response of noninitiated and initiated primary rat tracheal epithelial (RTE) cell cultures to the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The endpoints measured were number of cells per culture, colony-forming efficiency, subculturability, and colony formation in soft agarose. Primary RTE cell cultures were exposed on Day 1 to either 0.2% dimethyl sulfoxide, or to 0.1 micrograms per ml of N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). Thereafter, the same cultures were exposed twice weekly from Days 6 to 30 to either 0.2% dimethyl sulfoxide or to TPA (10 pg/ml). Sequential exposure to MNNG and TPA did not increase the number of viable cells per culture beyond that seen in MNNG-exposed cultures. Determination of the frequency of colony-forming cells 10 days after the end of the initiation-promotion treatment (Day 40 of culture) revealed a marked enhancement in colony-forming efficiency of treated cultures compared to dimethyl sulfoxide-exposed control cultures. However, sequential exposure to MNNG and TPA had an additive or slightly more than additive effect on the colony-forming efficiency of RTE cells exposed to MNNG or TPA only. Treatment of the primary cultures with MNNG alone or TPA alone increased the subculturability of RTE cells to a similar extent. The sequential exposure to MNNG followed by TPA appeared to have an additive effect on the frequency of subculturability. The most pronounced effect of the sequential MNNG-TPA exposure as compared to single-agent exposure was a marked enhancement of the anchorage-independent (ag+) phenotype. Of the cultures treated with MNNG followed by TPA, over 50% were ag+ at 60 days. In contrast, of the cultures treated either with MNNG alone or with TPA alone, only 3% were ag+ on Day 60. (All control cultures were ag-.) Colony-forming efficiency in soft agarose also increased disproportionately between 60 and 120 days in initiated-promoted cultures. These experiments indicate that the major effect of the tumor promoter TPA on initiated RTE cell cultures is to enhance the appearance of the late ag+-phenotype.