Serum levels of four cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-10 (IL-10)] were measured in nine diurnally active healthy adult male subjects at 3-h intervals during a 24-h period. Statistical evaluation by analysis of variance and/or the least- squares fit of a cosine model revealed significant 24-h rhythms for each cytokine. Although the amount of IL-2 in the serum was low, the levels fluctuated to form a single peak at approximately noon. In contrast, the other three cytokines exhibited a biphasic temporal pattern. In subjects with detectable TNF-alpha levels, the first peak occurred at 07:30 and the second at 13:30. IL-10 levels also exhibited a biphasic pattern, with one peak at 07:30 and the second 12 h later at 19:30. GM-CSF levels were last to rise, first peaking at approximately 13:30 and then again at 19:30. These results suggest temporal patterns that are unique for each cytokine, generally with daytime highs and nighttime lows.