This study was designed to investigate the effects of moderate-intensity endurance training on basal natural killer (NK) cell cytolytic activity in murine splenocytes that were enriched for 1) NK1.1+ cells or 2) macrophages and NK1.1+ cells. Mice were assigned to sedentary (Sed), treadmill control (TM), or treadmill-trained (Trn) groups. Splenocyte number, the percentages of NK1.1+, large granular lymphocytes (NK1.1+, LGL-1+), and other subpopulations did not change in Trn mice. Approximately 70% of cells enriched for NK1.1+ expressed this surface antigen. Lytic units (LU) expressed per LGL-1+ cell were significantly lower in Trn [83.9 +/- 3.2 (SE)] compared with Sed (109.5 +/- 7.5) and TM (101.3 +/- 6.4) groups. When macrophages remained in the in vitro assay, LU per LGL-1(+) cell did not differ across groups. The results indicate that highly enriched NK1.1+ cells from Trn mice had lower NK cell activity compared with Sed mice. No differences in NK cell activity were observed when cells were enriched for NK1.1+ cells and macrophages. These findings support the hypothesis that macrophage modulation of NK cells may be one mechanism contributing to augmented basal NK cell activity in endurance-trained individuals.