In order to quantify human interleukin-8 (IL-8), which is chemotactic for T cells and basophils as well as neutrophils, we developed an enzyme-linked immunosorbent assay (ELISA). Since binding inhibition tests indicated that three monoclonal antibodies (mAbs; BS-1, WS-4, WS-6) blocked the binding of 125I-labelled IL-8 to neutrophils, we tested an ELISA using these mAbs as primary antibodies, rabbit anti-IL-8 Ab as the secondary antibody, and alkaline phosphatase-labelled goat anti-rabbit Ab as the conjugate. Among the three mAbs tested, WS-4 was the most sensitive with a detection limit of 16 pg/ml. Several other cytokines, including monocyte chemotactic and activating factor (MCAF), which is structurally related to IL-8, showed no cross-reactivity in this system, indicating that this ELISA is specific for IL-8. The coefficients of variation for the intra- and interassays were below 10%. Furthermore, this ELISA also detected natural IL-8 (including both 72 and 77 amino acid forms) produced by cultured human cells and cell lines stimulated with IL-1, suggesting that this system will be useful in the detection of natural IL-8 in various body fluids.