Recently we have purified four neutrophil chemotactic factors from conditioned medium of rat granulation tissue. Two chemokines besides cytokine-induced neutrophil chemoattractant-1 (CINC-1, formerly called CINC) and CNC-3/macrophage inflammatory protein-2 (MIP-2) were novel chemoattractants, designated as CINC-2 alpha and CINC-2 beta. In the present report, we developed an enzyme-linked immunosorbent assay (ELISA) specific for CINC-2. The biotin-streptavidin sandwich ELISA for CINC-2 beta detected CINC-2 alpha and CINC- 2 beta equally well between 1 ug/mI and 300 ng/ml, but did not show cross-reactivity with CINC-1 and CINC-3. The concentrations of CINC-1 and CINC-2 in the exudate during rat lipopolysaccharide (LPS)-induced inflammation were determined. The CINC-1 concentration in the exudate reached a maximum at 4 h after LPS injection, whereas the CINC-2 level steadily increased up to 8 h. The number of infiltrated cells in the exudate increased linearly until 6 h and gradually up to 8 h. Increase in the cell number was correlated with total concentrations of CINC-1 and CINC-2. The results suggest that CINC-2 as well as CINC-1 plays an important role in accumulation of neutrophils into the inflammatory lesion of LPS- induced inflammation in rats.