Central release of Crh has recently been implicated in modulating natural killer cell (NK) activity independent of its role in activation of the hypothalamic-pituitaryadrenal axis. In the present study, NK- and interleukin-2 (IL- 2)-activated NK cytotoxicity against K-562 target cells was examined in porcine lymphocytes cultured in vitro with ACTH or pig lymphocytes after iv or im ACTH administration. Physiological concentrations of porcine ACTH (10-8-10-11 M) added to the culture medium had no direct influence on NK- or IL-2- stimulated NK cytotoxicity. In a second experiment four unrestrained pigs with indwelling catheters given an iv bolus of vehicle or ACTH (1 IU/kg BW) at 0800 h showed significantly elevated cortisol levels for 3 h after ACTH. Although serum cortisol had returned to baseline by 4 h after ACTH treatment, NK- and IL-2-stimulated NK cytotoxicity was dramatically elevated (P < 0.01) compared to that in saline-injected controls. NK cytotoxicity in control pigs followed a diurnal pattern, with low morning and high evening cytotoxicity. Exogenous ACTH, given by bolus in the morning, prevented the normal morning decline in NK cytotoxicity. Because of this unexpected dramatic increase in NK- and IL-2-stimulated NK cytotoxicity in animals given ACTH, the experiment was replicated in two subsequentstudies using 16 pigs (8 controls and 8 experimental) in each. Pigs were injected im with either ACTH (1 Iu/kg Bw) or an equivalent volume of saline at 0600 h. Two hours later, blood was collected by venipuncture to determine NK cytotoxicity and measure the cortisol response. As was observed in the previous study, NK- and IL-2-stimulated NK cytotoxicity was significantly greater (P < 0.01) than that in saline-injected controls. In the final experiment pigs were given either ACTH (1 Iu/kg Bw) or an equivalent volume of saline at 1800 h. Two hours later, blood was collected by venipuncture to determine NK cytotoxicity and cortisol response. ACTH administered in the evening increased NK cytotoxicity, but the effect was only marginally significant and far less dramatic than in previous studies. Because ACTH had little effect on NK- and IL-2-stimulated NK activity in vitro, we hypothesize that the stimulatory effect of exogenous ACTH is mediated through an indirect mechanism, possibly through the suppression of central Crh as a result of elevated cortisol. This effect is more pronounced when the stimulatory dose of ACTH is given at a time in the circadian cycle when NK cytotoxicity is normally low.