The estradiol (E2) to estriol (E3) ratio during human pregnancy depends on fetal liver hydroxylation of fetal adrenal dehydroepiandrosterone sulfate (DHEAS) and conversion by the trophoblast of DHEAS and 16 alpha-hydroxy-DHEAS (16 OH-DHEAS) to estrone (E1), estradiol (E2), and estriol (E3), respectively. It is not known whether the conversion of DHEAS into E1 and E2 influence the conversion of 16OH-DHEAS into E3 and vice versa. To examine this question, we studied these interactions in human choriocarcinoma JEG-3 cells. In serum-free medium (Dulbecco's Modified Eagle's Medium), JEG-3 cells secreted hCG [27 +/- 3 (+/- SEM) ng/mg cellular protein X 24 h] and progesterone (22 +/- 2.5), but neither C-19 nor C-18 steroids. The addition of DHEAS resulted in secretion of E1 and E2; at a concentration of 500 ng DHEAS/ml, the secretion of E1 (1 +/- 0.16) and E2 (11 +/- 3.1) was maximal, while E3 remained undetectable. The addition of 1000 ng 16 OH-DHEAS/ml resulted in maximum E3 secretion (13 +/- 1.8), while E1 and E2 remained undetectable. The addition of increasing concentrations of DHEAS to cultures exposed to 1000 ng 16OH-DHEAS/ml caused a decrease in E3 secretion and increased secretion of E1 and E2. Conversely, addition of increasing concentrations of 16OH-DHEAS in cultures exposed to 500 ng DHEAS/ml resulted in inhibition of E1 and E2 secretion and increased E3 secretion. A concentration of 16OH-DHEAS that inhibited the conversion of DHEAS into E1 and E2 neither altered the intracellular to extracellular steroid ratios (approximately 0.1) nor reduced the secretion of DHEA, androstenedione, and testosterone. The inhibitory effect of 16OH-DHEAS was minimal at low DHEAS concentrations (favoring the secretion of E1 and E2) and was greatly enhanced at concentrations of DHEAS that induced maximum E1 and E2 secretions. The results indicate that in trophoblastic cells, the metabolism of DHEAS can modulate E3 secretion, and the metabolism of 16OH-DHEAS can modulate the secretion of E1 and E2; and this regulatory mechanism appears to take place at the level of the aromatase system.