Male C3He mice were trained to run on a treadmill (final speed, slope, and duration of 30 m/min, 8 degrees, 30 min/day, 5 days/week, respectively) for 10 weeks or they remained sedentary. At the end of the training program, half of the mice were sacrificed and half were given a single bout of exercise to exhaustion (50% stepwise increases in final running speed for 2-min intervals). Splenic catecholamine concentrations, splenic natural killer cell cytolytic activity against YAC-1 tumor targets, and frequency of asialo GM1 (a murine natural killer cell surface glycolipid)-positive splenocytes were assessed. Exhaustive exercise in both trained and untrained mice reduced the in vitro killing of tumor targets by splenic natural killer cells relative to killing by splenocytes from mice which did not undergo the acute exercise bout (P less than 0.05). The frequency of asialo GM1-positive splenocytes was also reduced in the exhaustively exercised animals (P less than 0.05). Training alone, without the additional stress of exhaustive exercise, reduced the frequency of asialo GM1-positive splenocytes relative to a sedentary condition (P less than 0.05), but did not compromise natural killer cell cytolytic activity against the tumor targets. Splenic epinephrine concentrations in the exhaustively exercised animals were elevated 3- to 5-fold above the concentrations observed in trained and sedentary mice. These results suggest that a single, acute exercise bout reduces the capacity of splenic natural killer cells to kill tumor targets in vitro and that training enhances splenic natural killer cell cytolytic activity, on a per cell basis, against tumor targets.