Ten experienced marathoners were exercised 3 h in the laboratory. Blood samples were collected at 0 h baseline, 1 h exercise, and 5 min, 1.5 h, 6 h, and 21 h recovery and were analyzed for total number of lymphocytes expressing membrane antigens found on natural killer (NK) cells. NK activity was also measured. Four of the seven subpopulations of lymphocytes studied, Leu-11+19+, Leu-11+19-, Leu-11+7-, and Leu-19+11-, showed significant within-subject effects over time, using repeated measures ANOVA. Simple contrasts with baseline values showed that, at 1.5 h and 21 h recovery, total number of lymphocytes bearing three different combinations of NK markers, Leu-11+19+, Leu-11+19-, and Leu-11+7-, were significantly decreased when compared with baseline values. At 1.5 h recovery, NK activity was significantly decreased below baseline levels for four of the six effector NK cell/target K562 myelogenous leukemia cell (E:T) ratios tested. At 6 h recovery, NK activity was still decreased significantly with the 12.5:1 and 3:1 E:T ratios. By 21 h recovery, NK activity did not differ significantly from baseline levels. Cortisol levels at 5 min post-exercise were negatively correlated with NK activity at 1.5 h recovery (r = -0.62, P = 0.05, 50:1 E:T ratio; r = -0.66, P = 0.04, 25:1 E:T ratio). Further research is needed to elucidate the effect these changes have on host immunosurveillance and immunoresponsiveness in vivo.