We examined the effect of restraint on testicular interstitial fluid (TIF) concentrations of ACTH, beta-endorphin-lipotropin (beta-E-LI) and testosterone and correlated those changes with plasma concentrations of ACTH, beta-E-LI, corticosterone, LH and testosterone in adult rats. Animals were subjected to 1, 2, or 3 h of restraint and were killed immediately following the stress period. Plasma values of ACTH and beta-E-LI were elevated above control values after 1 and 2 h, but not after 3 h of restraint. Plasma corticosterone showed a similar response to restraint except that concentrations were also elevated after 3 h. Plasma testosterone concentrations were elevated after 1 h of restraint, but after 3 h of restraint had fallen below control values. Restraint reduced plasma testosterone concentrations without altering plasma LH concentrations. The decline in plasma testosterone during restraint was associated with a parallel decrease in testosterone in the TIF. Concentrations of ACTH and beta-E-LI were 6- and 3-fold greater in TIF than in the plasma. While 1 or 2 h of restraint did not affect ACTH and beta-E-LI in TIF, values of these hormones were elevated in rats exposed to 3 h of restraint. These data, coupled with recent reports that testicular proopiomelanocortin (POMC)-derived peptides may modulate testicular steroidogenesis, suggest that these factors may play an autocrine or paracrine role in mediating stressor-induced changes in testicular function.