Insulin administration to overnight fasted rats causes a dose-dependent decline in plasma glucose concentrations and a dose-dependent increase in plasma ACTH concentrations. The ACTH response, but not the glucose response, was blocked by treatment with chlorpromazine-morphine-pentobarbital, indicating that the main factors triggering the ACTH response are of central, rather than peripheral, origin. To study whether insulin affected the turnover of CRF and vasopressin (AVP) in the zona externa of the median eminence (ZEME), we determined the rate of decline of both hypophysiotropic factors in rats with or without blockade of axonal transport by colchicine. In the ZEME, the concentrations of CRF and AVP were assessed by quantitative immunocytochemistry (QICC) in tissue sections or by RIA in median eminence extracts. QICC allows selective quantification of AVP and other peptides within the ZEME. The changes in the CRF content, as measured by QICC and RIA, were linearly correlated (r = 0.99), demonstrating that changes in peptide-staining intensity reflect changes in peptide content. Colchicine, when given intracisternally in a nontoxic dose of 5 micrograms, had no marked effect on resting plasma levels of ACTH and only slightly reduced the ACTH response to insulin-induced hypoglycemia. In the ZEME, CRF and AVP concentrations at rest were not affected by colchicine. In colchicine-treated rats insulin-induced hypoglycemia resulted in a prominent decline in CRF and AVP concentrations in the ZEME. The CRF concentration declined at a rate of 23%/h over a period of 3 h. The AVP concentration declined to a similar extent as CRF over the first hour, but tended to fall at the later time points. We conclude that hypoglycemia increases turnover of both CRF and AVP in the ZEME. However, the turnover rates of both hypophysiotropic peptides do not appear to be quantitatively coupled.