17 alpha-Ethynylestradiol is extensively sulfated but the sulfate is thought to primarily be a storage form of this estrogen. 2-Hydroxylation is clearly the major oxidative reaction, and the 2-hydroxy derivative is further transformed by methylation and glucuronidation prior to urinary and fecal excretion. Alterations in the rate of 2-hydroxylation can have major effects on the pharmacokinetics and effectiveness of 17 alpha-ethynylestradiol as a contraceptive. The major human catalyst of the 2-hydroxylation reaction is liver microsomal cytochrome P-450 IIIA4. Lesser amounts of this enzyme are found in other tissues such as the intestine and may contribute to overall clearance of the orally administered contraceptive. In individuals with very low amounts of this enzyme other forms of cytochrome P-450 may make some contribution. Levels of cytochrome P-450 IIIA4 vary widely among individuals and can explain the variation in rates of 17 alpha-ethynylestradiol 2-hydroxylation. The known inducibility of the enzyme by barbiturates and rifampicin explains their effects in enhancing 17 alpha-ethynylestradiol clearance and reducing the effectiveness of the drug. Mechanism-based inactivation of cytochrome P-450 IIIA4 can be seen with 17 alpha-ethynylestradiol and other 17 alpha-acetylenic steroids, and the progestogen gestodene appears to be unusually active in this regard. Other unknown factors may also modulate levels of cytochrome P-450 IIIA4 and its ability to catalyze 17 alpha-ethynylestradiol 2-hydroxylation.