A highly sensitive time-resolved fluoroimmunoassay of human plasma cytokines is described. The cytokines such as interleukin-1α (IL-1α), tumor necrosis factor α (TNF α) are known to be acute inflammatory cytokines and it has been reported that these cytokines are secreted into blood by physical exercise. In this study, a sandwich-type immunoassay of cytokines was established using a europium chelate BHHCT-Eu³⁺ as a powerful labeling material. The minimum detection limits of cytokines, i.e. IL-1α, TNF α, and interferon γ (IFN γ) were about 1/10 smaller than those of enzymelinked immunosorbent assay currently used. By this immunoassay we investigated cytokine increase/decrease in plasma which was thought to derive from the myocytes damaged by bicycle exercise. Healthy young men performed two kinds of bicycle ergometer exercises, under conditions of an incremental and a constant loading. Blood samples were taken before, during, and after exercises, and the concentration levels of plasma IL-1α, TNF α, and IFN γ were determined. In the case of incremental exercise, IL-1α increased significantly at the first stage but decreased to the basal level from the second stage, in spite of heavier exercise. In the case of 30 min constant exercise, the level of plasma IFN γ increased in recovery period, 2 h after the light-exercise. TNF α level was significantly higher in a heavy-exercise. The concentration of IL-1α peaked at the early stage of the incremental exercise; this fact has not been reported in previous studies. This cytokine is unique in showing a sudden increase during the early stage, while others increase after the exercise. Our highly sensitive assay made it possible to detect a slight change in plasma cytokines.