We have shown that fetal adrenal fetal zone (FZ) volume and serum dehydroepiandrosterone sulfate (DHAS) levels were increased, whereas definitive and transitional zone (DZ/TZ) volume was unaltered, in baboons in which estrogen levels were suppressed by the administration of the aromatase inhibitor letrozole. The interaction of the melanocortin 2 receptor (MC2R) with its accessory protein (MRAP) is essential for trafficking MC2R to the adrenal cell surface for binding to ACTH. The present study determined whether the estrogen-dependent regulation of fetal adrenocortical development is mediated by ACTH and/or expression/interaction of MC2R and MRAP. Fetal pituitary proopiomelanocortin mRNA and plasma ACTH levels and fetal adrenal MC2R-MRAP interaction were assessed in baboons in which estrogen was suppressed/restored by letrozole/letrozole plus estradiol administration during the second half of gestation. Although fetal pituitary proopiomelanocortin and plasma ACTH levels and fetal adrenal MC2R and MRAP protein levels were unaltered, MC2R-MRAP interaction was 2-fold greater (P < .05) in the DZ/TZ in letrozole-treated baboons than in untreated animals and restored by letrozole plus estradiol treatment. We propose that the increasing levels of estradiol with advancing pregnancy suppress interaction of MC2R with MRAP, thereby diminishing MC2R movement to the cell membrane in the DZ/TZ. This would be expected to reduce progenitor cell proliferation in the DZ and migration to the FZ, thereby restraining FZ growth and DHAS production to maintain fetal adrenal DHAS and placental estradiol levels in a physiological range late in gestation.