The effects of restraint stress applied at different times of the day on levels of five stress-responsive plasma hormones (ACTH, β-endorphin, β-LPH, corticosterone and prolactin) and pituitary cyclic AMP levels were assessed. Different groups of rats were subjected to 15 min of restraint stress at 2-hour intervals over a 24-hour period. Rats were sacrificed immediately upon removal from their home cage (controls) or immediately following restraint (stressed). The time of day of stress exposure markedly affected the stress responses measured. Generally, responses to stress applied at the beginning of the dark cycle (18.00) were less than those seen following stress applied at the beginning of the light cycle (06.00). Stress at 06.00 increased levels of pituitary cyclic AMP 10-fold, while stress applied at 18.00 did not significantly increase pituitary cyclic AMP levels. In stressed rats, high correlations were seen among levels of hormones derived from the common precursor, proopiomelanocortin (ACTH, β-endorphin, β-LPH) and between these hormones and levels of pituitary cyclic AMP. These findings support the hypothesis that pituitary cyclic AMP is involved in the stress-induced release or synthesis of the pituitary hormones ACTH, β-endorphin, and β-LPH.