Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PM phi) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PM phi or bone marrow-derived macrophages (BMM phi) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8(-/-)) and wild-type (WT) mice. We found that CCR8(-/-) PM phi demonstrated attenuated secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PM phi but not BMM phi. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PM phi aggregation. Similar to CCR8(-/-) PM phi, R243 attenuated secretion of TNF-alpha, IL-6, and most strikingly IL-10 from WT PM phi, but not BMM phi. CCR8(-/-)PM phi and R243-treated WT PM phi both showed suppressed c-jun N-terminal kinase activity and nuclear factor-kappa B signaling after LPS treatment when compared with WT PM phi. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8(-/-) mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMQ. Through use of CCR8(-/-) mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.