Bile acids have been implicated as tumor promoters that enhance epithelial proliferation and the development of colonic tumors. This study investigated the effects of bile acids on the growth of in vitro models of human colonic epithelial cells. Cell lines with varying degrees of differentiation (Caco2, HT29, LS174T, and Lovo) were studied. Cell viability and number were measured by a tetrazolium (MTT) spectrophotometric assay. Enhanced cell growth was not observed with any bile acid over the range 10 nmol/L to 2.5 mmol/L. Cytotoxicity was consistently observed at concentrations of unconjugated bile acids greater than 0.1 mmol/L The bile acid concentration at which 50% growth inhibition occurred was similar for all cell lines and increased in the following order: deoxycholic acid = chenodeoxycholic acid < taurodeoxycholic acid < ursodeoxycholic acid < taurochenodeoxycholic acid < cholic acid < tauroursodeoxycholic acid. Coincubation of tauroursodeoxycholic acid (TUDC) with taurodeoxycholic acid (TDC) or taurochenodeoxycholic acid (TDCD) reversed the short-term (30-minute) cytotoxicity and release of glycoprotein induced by TDC or TCDC regardless of differentiation status. In contrast, TUDC did not reverse the cytotoxicity of deoxycholic acid. Unconjugated ursodeoxycholic acid did not alter short-term cytotoxicity of any bile acid. These data indicate that bile acids do not stimulate cell growth in undifferentiated or differentiated colon cancer cell lines, in contrast to normal colonic epithelium in vivo. Bile acid cytotoxicity correlates with the relative hydrophobicity of the bile acid. Because tauroursodeoxycholic acid alters the cytotoxicity of hydrophobic bile acids in vitro, further understanding of bile acid interactions in the colon may have important implications in altering tumor promotion.