Primary and secondary bile acids such as cholic (CHA), deoxycholic (DCA) and lithocholic (LCA) acids have been shown to increase colon tumorigenesis. It has been suggested that inhibition of xenobiotic metabolizing enzymes such as glutathione S-transferase (GST) and UDP-glucuronyltransferase (UGT) by bile acids may be a factor in the development of colon cancer. While enzyme inhibition has been demonstrated in vitro, it is unclear whether feeding bile acids modulates colonic GST and UGT in vivo. To test this notion, male, Sprague-Dawley rats (n = 100) were assigned to a control (CON) or test diets containing 0.2% CHA, DCA, LCA or ursodeoxycholic acid (UDCA). After 5 weeks, colonic tissue was harvested and used for enzyme and cell proliferation measurements. The response to bile acids varied with the enzyme measured and appeared isoenzyme specific. GST-alpha activity was lower in the bile acid fed groups compared with CON. While GST-mu was lower in the LCA-fed group, GST-pi was lower in the DCA-, CHA- and UDCA-fed groups. Unlike GST, both UGT and NADPH-cytochrome P-450 reductase (CYC) activities were increased by bile acids. The proliferative response of the colonic epithelium varied with the bile acids and was regionally specific. These data demonstrate that feeding bile acids alters the activity of colonic phase I and II enzymes; however, the physiological effect of these enzymatic perturbations is yet to be determined.