The Multiple Intestinal Neoplasia (Min) mouse carries the murine homologue of the human APC gene. To overcome the difficulties of culturing normal colonic mucosal cells in vitro we developed an F1 hybrid mouse ("Immortomouse" (H-2Kb-tsA58 SV40 large T)x Min) which carries both the Min mutation and a temperature-sensitive mutant of the SV40 large T gene. We have derived epithelial cell lines from both isolated colonic crypts and the liver of this hybrid mouse. The colonic epithelial cultures are only conditionally immortalized and growth ceases at the non-permissive temperature indicating that these cells have retained their normal phenotype. Fibroblast cultures have also been obtained from both small intestinal stroma and skin. The polymerase chain reaction (PCR) assays indicated that the cell lines all contain both the Min mutation and the SV40 large T gene. This study demonstrates the usefulness of the "Immortomouse" for developing cultures from tissues such as the intestine that have previously proved very difficult to culture in vitro.