This study was designed to test whether a single 50-mg dose of the opioid antagonist naltrexone hydrochloride, ingested 60 min before 2 h of moderate-intensity exercise (i.e., 65% peak O2 consumption), influenced the exercise-induced augmentation of peripheral blood natural killer cell cytolytic activity (NKCA). Ten healthy male subjects were tested on four occasions separated by intervals of at least 14 days. A rested-state control trial was followed by three double-blind exercise trials [placebo (P), naltrexone (N), and indomethacin] arranged according to a random block design. The indomethacin exercise trial is discussed elsewhere (S. G. Rhind, G. A. Gannon, P. N. Shek, and R. J. Shepherd. Med. Sci. Sports Exerc. 30: S20, 1998). For both the P and N trials, plasma levels of beta-endorphin were increased (P < 0.05) at 90 and 120 min of exercise but returned to resting (preexercise) levels 2 h postexercise. CD3(-)CD16(+)CD56(+) NK cell counts and NKCA were significantly (P < 0.05) elevated at each 30-min interval of exercise compared with correspondingly timed resting control values. However, there were no differences in NK cell counts or NKCA between P and N trials at any time point during the two trials. Changes in NKCA reflected mainly changes in NK cell count (r = 0.72; P < 0.001). The results do not support the hypothesis that the enhancement of NKCA during prolonged submaximal aerobic exercise is mediated by beta-endorphin.