To investigate the drug-metabolizing potential of different sub-populations of cells along the villus-crypt surface of the small intestine, the major monooxygenase activities directed towards the substrates benzo[a]pyrene (BP), 7-ethoxycoumarin and ethylmorphine were studied. The cells were isolated in sequential fractions corresponding to the villus tip-to-crypt gradient in the small intestinal epithelium of the rat. Cells from the upper- and mid-villus regions were rich in aryl hydrocarbon (BP)hydroxylase (AHH) and 7-ethoxycoumarin deethylase (7-ECDE) activities whereas in crypt cells the activities of these enzymes were at the level of detectability. Ethylmorphine demethylase (EMD) was not detectable in the entire villus-crypt surface. The intestinal epithelial cells responded strongly to inducers. 3-Methylcholanthrene (3-MC), given to rats 24 hr previously, induced increases in AHH activity of 4- to 7-fold in the villus and of 19- to 26-fold in the crypt cells. 7-ECDE had a similar pattern. The induced level of monooxygenase activity in crypt cells was sustained for a longer time, followed in order by consecutively higher cells of the villus. Phenobarbital caused maximal expression of EMD activity in the mid-villus region whereas the activity in crypt cells was half the maximal activity. PB also significantly induced AHH and 7-ECDE in the intestinal epithelium. 7,8-Benzoflavone inhibited AHH activity to the same degree in all the cell fractions. The apparent Km for AHH was 5 microM (BP). Treatment of rats with 3-MC, after 24 hr, enhanced the Km and Vmax differently in the cells along the villus-crypt surface. The Km value in the villus region increased, whereas it did not change in the crypt cells; Vmax increased 6-fold in the villus and about 12-fold in the crypt cells, above their basal levels. The results suggest that the intestinal cells are capable of biotransforming various xenobiotics. The higher sensitivity of their monooxygenases, particularly of the crypt cells, might protect them directly or render the cells capable of generating metabolites that aid and abet toxicity toward target tissue in vivo.