The changes in the hypothalamic content of corticotrophin-releasing factor (CRF), the plasma concentrations of ACTH and corticosterone were studied in the intact male rat. CRF activity was estimated by bioassay and plasma ACTH levels by radioimmunoassay. The variation in CRF hypothalamic content reflected CRF secretion since plasma ACTH and corticosterone increased concomitantly. The activity of CRF separated into 2 peaks, one occurring immediately (2 min) and a second about 80 min later after the start of stress. The elevation of plasma ACTH and corticosterone followed the first CRF activity peak, and both showed a sustained time course. Cycloheximide pre-treatment (1 mg/100 g ip) was used to test whether protein synthesis was required for secretion of CRF. Treatment with cycloheximide reduced the incorporation of [14C]L-alanine into hypothalamic and pituitary proteins. The second CRF activity peak was completely abolished while the first activity peak was essentially intact. Plasma ACTH increased in response to the first CRF activity peak, but then declined. Plasma corticosterone remained low throughout, possibly as a consequence of inhibition of adrenal protein synthesis. When a second stress stimulus (fracturing of one leg without anaesthesia) was superimposed, CRF activity and plasma ACTH did not show any incremental response. These results show that the second CRF activity peak is sensitive to cycloheximide inhibition of protein synthesis, while the first activity peak appears to be cycloheximide resistant.