The cytokines IL-1 and TNFα have been shown to upregulate the transcription of matrix metalloproteinases (MMPs) as well as down regulate the synthesis of matrix components, including aggrecan and collagen type II. These cytokines are elevated in joints of patients with inflammatory arthritis and, by modulating both MMP and matrix biosynthesis, may contribute to the ultimate destruction of articular cartilage in these diseases. Very little is currently known about the signaling pathways through which these cytokines modulate chondrocyte gene expression. In fibroblasts, monocytes and T-cells, IL-1 and TNF-α appear to modulate gene expression through a pathway involving the nuclear translocation of NFκBp65. SV-40 immortalized human chondrocyte cell lines have recently been described in which aggrecan and collagen type II gene expression can be inhibited by IL-1α, under conditions shown to inhibit matrix gene expression in primary chondrocyte cultures. Using a similar immortalized human chondrocyte line, we have evaluated the translocation of NFκBp65 and the degradation of its native inhibitor IκB-α in cells stimulated by IL-1α, IL-1β or TNFα. In unstimulated cells, NFκBp65 is localized throughout the cytoplasm and can be detected in cytosolic extracts along with intact IκB-α. All three cytokines stimulate the translocation of NFκBp65 from the cytoplasm to the nucleus, resulting from degradation of cytosolic IκB-α. There is quantitative degradation of IκB-α after 20 minutes of treatment with all three cytokines at concentrations of 100 pM or greater. Within 2 hours the IκB-α levels return to the baseline levels detected in unstimulated cells Concentrations of 10 pM, or lower, result in virtually no degradation of IκB-α. This is the first definitive report to show that the signaling by IL-1α, IL-1β and TNFα is mediated through the NFκBp65/IκB-α pathway in human chondrocytes. Examination of the relationship of this signaling pathway with expression of enzymes and matrix components may allow for identification of unique targets to modulate chondrocyte matrix degradation and/or biosynthesis in inflammatory joint disease.