Although it has been recognized for over a decade that hypothalamic-pituitary disconnection (HPD) in the fetal sheep prevents the late gestation rise in plasma cortisol concentrations, the underlying mechanisms remain unclear. We hypothesized that reductions in adrenal responsiveness and ACTH receptor (ACTH-R) expression may be mediating factors. HPD or sham surgery was performed at 120 days of gestational (dGA), and catheters were placed for blood sampling. At around 138 dGA, fetuses were killed, and adrenals removed for cell culture and analysis of ACTH-R mRNA and protein. After 48h adrenocortical cells were stimulated with ACTH for 2h, and the medium collected for cortisol measurement. The same cells were incubated overnight with medium or medium containing ACTH or forskolin (FSK), followed by ACTH stimulation (as above) and cortisol and cellular ACTH-R mRNA analysis. HPD prevented the late gestation increase in plasma cortisol and bioactive ACTH, and reduced adrenal ACTH-R mRNA and protein levels by over 35%. HPD cells secreted significantly less cortisol than sham cells (3.2±1.2 ng/ml/2h vs. 47.3±11.1 ng/ml/2h) following the initial ACTH stimulation. Overnight incubation of HPD cells with ACTH or FSK restored cortisol responses to acute stimulation to levels seen in sham cells initially. ACTH-R mRNA levels in cells isolated from HPD fetuses were decreased by over 60%, while overnight incubation with ACTH or FSK increased levels by approximately 2 fold. Our findings indicate that the absence of the cortisol surge in HPD fetuses is a consequence, at least in part, of decreased ACTH-R expression and adrenal responsiveness.