The effect of 45 min of high- (80% VO2max) vs moderate- (50% VO2max) intensity treadmill exercise on natural killer cell cytotoxic activity (NKCA) was investigated in 10 well-conditioned (66.0 +/- 1.9 ml.kg-1.min-1), young males (22.1 +/- 1.3 yr). Blood samples were taken before and immediately after exercise, with three more samples taken during 3.5 h of recovery, and analyzed for proportion of NK cells (CD3-CD16+CD56+) and NKCA. Exercise at 80% vs 50% VO2max resulted in a greater immediate postexercise increase in proportion of NK cells, followed by a 1-h and 2-h decrease below preexercise levels for both intensity conditions. NKCA rose significantly above preexercise levels following high- but not moderate-intensity exercise. For both exercise intensity conditions, NKCA tended to drop below preexercise levels by 1 h postexercise, rising back to preexercise levels by 3.5 h postexercise. When NKCA was expressed on a per-NK cell basis, however, no change relative to preexercise levels occurred following moderate-intensity exercise, while a significant increase occurred after 2-h recovery from high-intensity exercise. These data demonstrate that both high- and moderate-intensity exercise are associated with significant shifts in circulating proportions of NK cells which significantly influence interpretation of NKCA data based on assays using separated mononuclear cells.