A novel, sensitive (0.5 ng) assay for acetaminophen, using HPLC with selective electro-chemical detection, enabled us to study rat small intestinal O-deethylation of phenacetin and compare it with corresponding 7-ethoxycoumarin- O-deethylation. Two in vitro systems, i.e. isolated intestinal mucosal cells and microsomal fractions thereof, were used to study kinetics for the O-deethylation of both substrates. K m app - and V max -values are similar for 7-ethoxycoumarin- and phenacetin- O-deethylation. Apparent K m -values varied between 50 and 70 μM in control rats and decreased after 3-methylcholanthrene pretreatment to 20–45 μM. V max -values were increased by 3-methylcholanthrene pretreatment. O-Deethylation was inhibited equally in cells and microsomes by α-naphtoflavone, but is inhibited more markedly in intestinal preparations after pretreatment with 3-methylcholanthrene. It is suggested that 7-ethoxycoumarin and phenacetin are O-deethylated by different forms of cytochrome P-450 with almost identical K m app and that these enzymes have a different distribution along the villus.