The present study was designed to explain the mechanism of the post-exercise down-regulation of human natural killer (NK) cell activity recently described by us. Fifteen young, healthy volunteers underwent 60 min of bicycle exercise at 75% of maximal oxygen uptake (VO2max). Six of the volunteers were exercised twice with an interval of at least one month. At the second exercise test they received oral indomethacin. Blood samples were collected before and during the last minutes of exercise as well as 2 h and 24 h after work. The NK cell activity (lysis fixed number of mononuclear cells) increased during bicycle exercise, dropped to a minimum 2 h later and returned to pre-exercise levels within 24 h. During bicycle exercise the percentage of NK cells (CD16+ cells) of mononuclear cells increased significantly but returned to normal within 2 h after exercise. Two hours after exercise, however, increased monocyte cell count and neutrophils were found. The in vitro release of prostaglandin E2 from mononuclear cells was increased. Furthermore, the neutrophil chemiluminescence response was also increased in the 2 h post-exercise period; this response is associated with prostaglandin E2 production by neutrophils. Indomethacin, whether administered in vivo or in vitro, fully restored the suppressed post-exercise NK cell activity. Finally, the NK cell activity of monocyte depleted mononuclear cells did not decrease below basal levels after exercise. These findings strongly indicate that prostaglandins released from monocytes and neutrophils are involved in the post-exercise down-regulation of NK cells.