Methadone and buprenorphine (BUP) are used for treatment of the pregnant opiate addict. CYP19/aromatase is the major placental enzyme responsible for the metabolism of methadone to 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and BUP to norbuprenorphine (norBUP). The aim of this investigation was to determine the effects of methadone and BUP on the activity of placental microsomal aromatase in the conversion of its endogenous substrates testosterone to 17β-estradiol (E 2) and 16α-hydroxytestosterone (16-OHT) to estriol (E 3). The conversion of testosterone and 16-OHT by human placental microsomes exhibited saturation kinetics, and the apparent K m values were 0.2 ± 1 and 6 ± 3 μM, respectively. V max values for E 2 and E 3 formation were 70 ± 16 and 28 ± 10 pmol/mg protein min, respectively. Also, data obtained revealed that methadone and BUP are competitive inhibitors of testosterone conversion to E 2 and 16-OHT to E 3. The K i for methadone inhibition of E 2 and E 3 formation were 393 ± 144 and 53 ± 28 μM, respectively, and for BUP the K i was 36 ± 9 and 6 ± 1 μM. The higher potency of the two opiates and their metabolites in inhibiting E 3 formation is in agreement with the lower affinity of 16-OHT than testosterone to aromatase. Moreover, the metabolites EDDP and norBUP were weaker inhibitors of aromatase than their parent compounds. The determined inhibition constants of methadone and BUP for E 3 formation by a cDNA-expressed CYP19 preparation were similar to those for placental microsomes. Therefore, data reported here suggest that methadone, BUP, and their metabolites are inhibitors of androgen aromatization in the placental biosynthesis of estrogens.