Using an organ culture/cell culture system, we transformed rat tracheal epithelial cells in vitro by exposure to MNNG. Ten tracheal organ cultures per group were exposed twice (at days 3 and 6) to 0,0.001, 1.0 or 10.0 microgram MNNG/ml of medium. Following this exposure, the explants were placed on the bottom of culture dishes to initiate epithelial cell outgrowths and establish primary cultures. Each explant was replanted 8-10 times to produce multiple outgrowths. The number of primary cultures and the number of subsequently established cell lines obtained was carcinogen-dose-dependent. The average number of primary epithelial cell cultures per explant after exposure to 0, 0.001 1.0 and 10.0 microgram MNNG/ml was 1.3, 1.5, 3.3, and 4.6, respectively. The average yield of cell lines per explant in these groups was 0, 0.8, 1.3, and 2.0, respectively. It is apparent that cell lines could only be established from carcinogen-exposed epithelium. These cell lines are currently being tested for tumorigenicity in vivo. To date, of 35 cell lines tested between the 5th and 15th passages, 5 cell lines from the 10 microgram MNNG/ml group and 2 from the 1.0 microgram MNNG/ml group have produced palpable tumors upon injection into immunosuppressed recipients.