IL-32 is a recently discovered cytokine that induces TNFα, IL-1β, IL-6, and chemokines. We investigated whether IL-32 is expressed in the synovia of patients with rheumatoid arthritis (RA) and studied associations with disease severity and the presence of other cytokines. Immunohistochemistry revealed that IL-32 is highly expressed in RA synovial tissue biopsies, whereas IL-32 was not observed in synovial tissues from patients with osteoarthritis. Moreover, in synovial biopsies from 29 RA patients with active disease, the level of IL-32 staining correlated with erythrocyte sedimentation rate, a marker of systemic inflammation (R = 0.63 and P < 0.0003). Synovial staining of IL-32 also correlated with indices of synovial inflammation (R = 0.80 and P < 0.0001) as well as synovial presence of TNFα (R = 0.68 and P < 0.004), IL-1β (R = 0.79 and P < 0.0001), and IL-18 (R = 0.82 and P < 0.001). IL-32 was a potent inducer of prostaglandin E₂ release in mouse macrophages and human blood monocytes, an important property for inflammation. After the injection of human IL-32γ into the knee joints of naïve mice, joint swelling, with pronounced influx of inflammatory cells and cartilage damage, was observed. In TNFα-deficient mice, IL-32-driven joint swelling was absent and cell influx was markedly reduced, but loss of proteoglycan was unaffected, suggesting that IL-32 activity is, in part, TNFα-dependent. IL-32, strongly associated with TNFα, IL-1β, and IL-18, appears to play a role in human RA and may be a novel target in autoimmune diseases.