Introduction: Interleukin-32 (IL-32) is a recently described cytokine that is a strong inducer of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, IL-1 beta, IL-6, and IL-8. The expression of this cytokine is highly increased in the rheumatoid synovium and correlated with the severity of joint inflammation. Little is known regarding the innate immune-related regulation of IL-32 by fibroblast-like synoviocytes (FLSs). We therefore investigated the effect of innate immune stimulation by ligands of Toll-like receptor (TLR)2, TLR3, and TLR4, and cytokines such as TNF-alpha and interferon (IFN)-gamma, on IL-32 expression by FLSs. Methods: FLSs were isolated from patients with rheumatoid arthritis (RA) according to the ACR criteria. Quantitative RT-PCR, confocal analysis, and ELISA were performed to evaluate IL-32 mRNA induction and IL-32 release by FLSs stimulated with TLR2 (BLP), TLR3 (poly I:C), and TLR4 (lipopolysaccharide) ligands, TNF-alpha and IFN-gamma. Results: TLR2, -3, and -4 ligands as well as IFN-gamma and TNF-alpha induced IL-32 beta,gamma and delta mRNA expression by RA FLSs. Mature IL-32 was expressed intracellularly and released by cells stimulated with the various activators. The IL-32 alpha isoform was expressed intracellularly in response to TNF-alpha and poly I:C and not released in culture supernatants. Stimulation of FLS with TNF-alpha, BLP, lipopolysaccharide, or poly I:C concomitant with IFN-gamma increased IL-32 expression compared with stimulation with IFN-gamma alone. Conclusions: IL-32 synthesis by FLSs is tightly regulated by innate immunity in rheumatoid arthritis. Thus TNF-alpha, IFN-gamma, double-strand RNA, hyaluronic acid, or other damage-associated molecular patterns (DAMPs), highly secreted in synovial tissues of RA patients, might trigger IL-32 secretion by FLSs. IL-32 might therefore represent a relevant therapeutic target in RA.