Two epithelial cell lines have been established from the duodenum (IEC-17) and the ileum (IEC-18) of outbred germfree Crl:CD(SD)GN rats. They have a very similar morphology and ultrastructure, a normal rat diploid karyotype, comparable growth rates, and a similar set of surface antigens as detected with monoclonal antibodies specific for intestinal epithelial cell surface proteins in vivo. Both cell lines do not grow in soft agar and when injected into syngeneic animals do not form tumors. The toxic effects of benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) were studied in these two cell lines. The IEC-18 cells were found to be much more sensitive to both chemicals than the IEC-17 cells. In particular, at concentrations greater than 1 microgram/ml, DMBA completely and irreversibly inhibited cell proliferation in the IEC-18 cells, whereas it produced a less marked and reversible inhibition in the IEC-17 cells. The metabolism of BP and DMBA to water-soluble products was studied; BP was apparently metabolized at similar rates in the two cell lines, whereas a striking difference was observed with DMBA, which was metabolized at a rate 10-15 times greater in the IEC-18 cells than that in the IEC-17 cells. These results suggest that cultured cell lines derived from different portions of the intestinal tract have similar properties in vitro, but they may have very different responses to chemical carcinogens.