Rheumatoid arthritis (RA) is chronic polyarthritis in which a variety of inflammatory cytokines play a role. Since tumor necrosis factor-alpha (TNF-alpha) is one of the most important cytokines in the pathogenesis of RA, we evaluated the feasibility of ribozymes as a therapeutic agent to control the inflammatory process of RA synovium. A hammerhead ribozyme against TNF-alpha was chemically modified to increase nuclease resistance and added to RA fibroblastlike cell cultures without using a delivery system. The cellular uptake of fluorescent-labeled ribozyme into synovial cells was found to last at least 48 hr by confocal laser scanning microscopy. The ribozyme targeting TNF-alpha gene inhibited both the expression of TNF-alpha mRNA and the secretion of TNF-alpha and IL-6. The cytotoxic effect by the ribozyme on synovial cells was negligible when determined by an alamar blue assay. Chemically modified ribozymes designed to suppress the TNF-alpha gene may be potential as a therapeutic agent for rheumatoid arthritis.