The cytokine tumor necrosis factor alpha (TNF-α) is a critical effector of the pathogenesis of rheumatoid arthritis (RA). We used oligonucleotide microarray (OM) analysis to assess TNF-α-modulated gene expression in cultured primary human synoviocytes in vitro. Genes identified include cytokines and inflammatory mediators, extracellular matrix and adhesion molecules, cell cycle and proliferation related proteins, transcription related proteins, and apoptotic mediators. OM identified 1185 differentially expressed genes in TNF-α-treated synoviocytes. The regulation of Nef-associated factor-1 (Naf1), an A20-binding, nuclear factor kappa B (NFκB) inhibitory protein was probed further given its putative role as an endogenous brake for the expression of some TNF-α-driven genes. Naf1 mRNA levels were higher in synovial biopsies from patients with active RA and seronegative arthropathy than in those from patients with osteoarthritis. These findings underscore the value of transcriptome analysis in cytokine-activated synoviocyte cultures in vitro as a means of identifying disease-associated genes in human arthritis, and implicate Naf1 as a potential modulator of TNF-α bioactivity in RA.