We examined the expression and participation of CCR6 and its ligand MIP-3alpha in rheumatoid arthritis (RA) by ELISA, RT-PCR, real-time PCR (TaqMan) analysis, monocyte chemotaxis, and two- and four-color flow cytometry. We found that RA synovial fluid (SF) contained significantly more MIP-3alpha than osteoarthritis (OA), indicating a potential role for MIP-3alpha in RA. IL-1beta, IL-18, and TNF-alpha stimulated RA fibroblast MIP-3alpha production at 48 hours of incubation in vitro. By TaqMan analysis, there were more CCR6 mRNA transcripts in RA synovial tissue (ST) than in OA or normal (NL) ST, and elevated MIP-3alpha mRNA expression in RA compared with NL ST. By FACS analysis, there were significantly elevated percentages of CD3+/CD4+/CD45RO+/CCR6+ memory lymphocytes found in RA peripheral blood (PB) compared with NL PB or RA SF. We also found that MIP-3alpha induced monocyte chemotactic activity at 1.25 pM, consistent with concentrations of MIP-3alpha found in RA SF. Furthermore, MIP-3alpha accounted for 40% of RA SF chemotactic activity for monocytes in modified Boyden chamber assays. We confirmed that MIP-3alpha may be binding a G-coupled protein receptor because in vitro monocyte chemotaxis was inhibited by preincubation of monocytes with pertussis toxin. RA patient clinical data revealed that CD3+ lymphocyte/CCR6 expression inversely correlated with the age of the patient, indicating that CCR6 expression may be important in the development of RA at a younger age. Overall, these studies indicate that MIP-3alpha and CCR6 may function to recruit monocytes and memory lymphocytes from the RA PB to the RA joint. These results further indicate that expression of CCR6, the receptor for MIP-3alpha, can be correlated with RA development.