The influence of alpha-naphthoflavone (ANF), rotenone, SKF 525-A, antimycin A and metyrapone on 7-ethoxyresorufin metabolism by rat small intestinal microsomes and epithelial cells was examined. ANF and rotenone were the most potent inhibitors of ethoxyresorufin (ER) deethylase activity in intestinal microsomes from control and 3-methylcholanthrene (MC)-treated rats. At a concentration of 0.05 muM, ANF inhibited approximately 85% of ER deethylase activity of intestinal microsomes from control animals and 55% of the activity of microsomes from MC-treated rats. The pH optimum for ER deethylation was 7.6, and the amounts of resorufin formed/mg protein/min for the uninhibited control and MC microsomes were 60 +/- 13 pmol and 1317 +/- 207 pmol, respectively. A 2.25 muM concentration of ANF inhibited approximately 55% of the ER deethylase activity associated with intestinal cells from MC-treated rats. 7-Ethoxycoumarin (EC) deethylase activity in intestinal microsomes had a pH optimum of 7.8, and was inhibited by ANF, rotenone, SKF 525-A and metyrapone in a manner similar to the results observed for ER deethylase. These results, in conjunction with previous investigations, suggest that rotenone and ANF may be most useful in differentiating between various species of cytochrome P-450.