Abstract The effect of diethylstilbestrol (DES), oestradiol (E 2), primaquine (PQ), chloroquine (CQ), 1-methylimidazole (1-MeI), metronidazole (MET) and antipyrine (AP) has been studied on rat liver microsomal metabolism of ethinyloestradiol (EE 2) by measuring the formation of 2-hydroxyethinyl-oestradiol (2-OHEE 2) using reverse phase high performance liquid chromatography. Using a substrate concentration of 25 μM, PQ, DES and E 2 produced the most marked effect with IC 50, values of 75, 100 and 100 μM respectively whereas CQ, MET and 1-MeI were less potent with IC 50, values of 335, 448 and 448 μM. AP inhibited EE 2 metabolism to only a small extent and an IC 50 value was not calculated. PQ (75 μM) inhibited the enzyme non-competitively decreasing the V max from 1.8 to 1.0 nmol/min/mg protein. E 2 (100 μM) inhibited the enzyme competitively with an increase in the K m from 17.9 to 55.6 μM. The results of this study indicate that steroidal and non-steroidal compounds have different affinities for EE 2 2-hydroxylase.