Trilostane, a competitive inhibitor of 3 beta-hydroxysteroid dehydrogenase, was administered intravenously to pregnant mares (n = 3) between day 277 and day 282 of gestation to determine its effect on the progestin milieu. In addition, placental tissue from mares at mid-gestation (150-300 days) (n = 4) were exposed to either deuterium-labelled pregnenolone alone or deuterium-labelled pregnenolone and trilostane to examine the effect of trilostane on placental metabolism of pregnenolone. Blood samples were collected from indwelling jugular catheters at frequent intervals for 48 h after infusion. Both plasma samples and incubation media were quantitatively analysed, after solid phase extraction and steroid derivitization, for concentrations of eight different progestin metabolites using gas chromatography and mass spectrometry. Trilostane infusion differentially affected the progestin milieu in vivo without inducing abortion. Forty-five minutes after infusion, maternal plasma concentrations of pregnenolone, 5-pregnene-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta,20 beta-diol and 3 beta-hydroxy-5 alpha-pregnan-20-one increased (P < 0.05) and remained high for 37 h. Concentrations of 5 alpha-pregnane-3,20-dione, 20 alpha-hydroxy-5 alpha-pregnan-3-one and 5 alpha-pregnane-3 beta,20 alpha-diol decreased 15 min after infusion (P < 0.05), yet 1.5 h after infusion, concentrations had increased and remained high until 37 h after infusion. Trilostane inhibited the conversion of pregnenolone to progesterone in vitro (P < 0.001) while mediating an increase (P < 0.05) in concentrations of 5 alpha-pregnane-3,20-dione and 3 beta-hydroxy-5 alpha-pregnan-20-one. These observations demonstrate that the pregnant mare possesses unique steroid hormone metabolic activity and suggests that another steroid, perhaps 5 alpha-pregnane-3,20-dione and not progesterone, is the important steroid precursor for the other progestin metabolites found in circulating plasma.