This study was designed to examine immunological responses to an acute bout of cycle ergometry exercise before and after moderate endurance training. Previously sedentary males were randomly assigned to matched training (n = 9) or control (n = 6) groups. Training comprised 12 weeks during which supervised cycle ergometer exercise took place [30 min at 65-70% of maximal oxygen intake (VO2max), 4-5 days .week-1]. An acute bout of exercise (60 min; 60% VO2max) was performed initially and after the 12-week interval. Samples of peripheral venous blood were taken at rest, after 30 and 60 min of exercise, and at 30 and 120 min post-exercise. Training improved VO2max by an average of 20% (40.6 to 49.2 ml.kg-1.min-1). Relative to baseline and control measures, the resting concentration of (CD3-CD16+/CD56+) natural killer (NK) cells increased by 22% (P < 0.05). The resting count of total CD25+ [interleukin-2 receptor (IL-2R) alpha chain] lymphocytes did not change following training, but dual staining analysis showed a 100% increase in the fraction of CD16+ CD25+ NK cells (P < 0.05). Likewise the resting CD122+ (IL-2R beta chain) lymphocyte count increased 35% after training, the greatest increases (44%) being in CD16+ CD122+ NK cells (P < 0.05). Soluble IL-2R levels also increased 33% (P < 0.05) after training. Following acute exercise at the same relative intensity, trained individuals exhibited a larger increase in the NK cell count, reduced lymphocytopenia, and attenuation of exercise-induced suppression of lymphocyte proliferation and IL-2 production (P < 0.05). In addition, smaller increases in CD4 and CD8 counts during exercise were noted, but with faster recovery post-exercise (P < 0.05). Addition of recombinant IL-2 (rIL-2) to phytohemagglutinin-stimulated peripheral blood mononuclear cell cultures did not reverse exercise-induced suppression of cell proliferation, either before or after training. However, rIL-2 did augment the spontaneous blastogenesis of exercise and post-training samples relative to baseline (P < 0.05). We conclude that moderate endurance training is associated with sustained alterations in immune function, both at rest and when exercising. Further investigations are necessary to determine the impact on overall health and susceptibility to disease.