Immunoreactive ACTH (I-ACTH) levels in the rat anterior pituitary and plasma, and immunoreactive corticotropin-releasing factor (I-CRF) concentrations in the median eminence (ME) and plasma were determined after adrenalectomy and in insulin-induced hypoglycemia. I-CRF was detected in plasma from normal rats (mean +/- SD, 5.6 +/- 0.9 pg/ml; n = 6). Gel filtration chromatography of I-CRF from pooled plasma of these rats revealed a single peak which eluted in the position of authentic rat CRF. I-CRF levels in ME and I-ACTH levels in anterior pituitary decreased immediately after adrenalectomy, then gradually increased to high levels 14 days after surgery. Plasma I-CRF and I-ACTH concentrations increased immediately after surgery, slightly decreased to near the control levels at 24 h, and then increased to high concentrations 14 days after surgery. Plasma and ME I-CRF levels 14 days after adrenalectomy, followed by daily dexamethasone replacement, were almost the same as control levels. In insulin-induced hypoglycemia, plasma I-ACTH and I-CRF concentrations increased and ME I-CRF content decreased at 30 and 60 min. These results suggest that plasma I-CRF levels reflect changes in hypothalamic CRF levels.