Loss of function of mismatch repair (MMR) genes underlies hereditary nonpolyposis colorectal cancer (HNPCC). However, the inability to maintain primary colon epithelial cells in culture has limited the analysis of the contribution of MMR gene defects to colorectal tumorigenesis. We have now established primary cultures of epithelial cells from the colon crypts of Msh2-/- p53-/- double-knockout mice. These cells undergo spontaneous transformation (soft agar colonies and s.c. tumor formation), with a progressively shorter latency as a function of increasing passages in culture. Treatment of early passage cells with the mutagen methylmethane thiosulfonate (MMS) further decreases the transformation latency of Msh2-/- p53-/- cells. Spontaneous transformation of p53-/- colonocytes is only observed using late passage cells, and methylmethane thiosulfonate-treated early passage p53-/- colonocytes do not form tumors when injected into immunodeficient mice. Together, these findings support the pathogenic role of MMR gene inactivation in colorectal tumorigenesis and provide an experimental model for the serial assessment of the molecular phenotype associated with Msh2 deficiency.