Rheumatoid arthritis (RA) is a chronic inflammatory disease of unknown etiology characterized by an infiltration of CD4+ T lymphocytes within the rheumatoid synovium. Cytokines have been shown to play a modulatory role in the pathogenesis of RA. We analyzed the expression of a T cell derived cytokine. interleukin 16 (IL-16), in relation to disease activity to characterize its biologic function in RA. Secreted IL-16 was measured by enzyme immunoassay in sera and synovial fluids (SF) from 25 patients with RA in comparison to 20 control samples from patients with osteoarthritis (OA). IL-16 expression in peripheral blood mononuclear cells (PBMC) was characterized by flow cytometric analysis after intracellular cytokine staining for IL-16. In synovial tissue specimens, IL-16 mRNA expression was analyzed by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). In parallel, expression of IL-16 was localized in synovial tissues by in situ hybridization and immunohistochemistry. Results were analyzed in relation to disease activity. IL-16 was detected at significantly higher levels in sera and SF of patients with RA in comparison to OA (p < 0.001). Flow cytometry of PBMC showed that a great proportion of both CD4+ and CD8+ T cells constitutively expressed the IL-16 protein. In synovial tissues, IL-16 mRNA levels were significantly elevated in comparison to OA controls (p < 0.001). In situ hybridization for IL-16 producing cells revealed a predominant accumulation of IL- 16 positive cells within the inflammatory infiltrates. A significant correlation between IL- 16 expression and local inflammatory activity could not be established (r = 0.27, p = 0.19) by microscopic analysis of the synovial cell infiltrate. In addition, no significant association was observed between serum, SF, and synovial tissue expression of IL-16 and clinical disease activity in RA. These data suggest IL-16 might play a role in the pathogenesis of chronic inflammation in RA. The lack of significant correlation between IL-16 expression, clinical disease activity, and local inflammatory activity suggests a regulatory rather than a proinflammatory function for IL-16 in the pathogenesis of chronic synovial inflammation in RA.