The opioid peptide methionine-enkephalin (Met-enkephalin) was measured in plasma and cerebrospinal fluid (CSF) of sheep in which the cisterna magna, carotid artery, and jugular vein were chronically cannulated. Venous blood plasma and CSF were collected before and after stress treatment and in control studies in conscious animals. Plasma and CSF were extracted with octadecylsilica and oxidized, and Met-enkephalin was measured as its Met-sulfoxide derivative by specific RIA. The molecular form of immunoreactive Met-enkephalin was characterized by peptide size exclusion chromatography of an octadecylsilica extract of sheep plasma through Bio-Gel P2, followed by reverse phase liquid chromatography, and was identical to Met-enkephalin and Met-sulfoxide-enkephalin. Insulin-induced hypoglycemia produced an elevation of plasma cortisol and an increase in the plasma concentration of Met-enkephalin. Acute hemorrhage led to an earlier and greater rise in plasma cortisol than that associated with insulin-induced hypoglycemia, but did not increase the concentration of Met-enkephalin in plasma. Neither form of acute stress increased the concentration of Met-enkephalin in CSF. These studies confirm that secretion of Met-enkephalin into blood can be dissociated from stimulation of the pituitary-adrenocortical system. They also show that circulating Met-enkephalin is elevated in conscious sheep during acute hypoglycemic stress, but plasma Met-enkephalin is unlikely to exert effects on the opiate receptors of periaqueductal or spinal nociceptive neurons under these conditions, since it does not enter cerebrospinal fluid in significant amounts.