Adenosine is an endogenous immunomodulator that has been shown to exhibit anti-inflammatory and immunosuppressive properties through a mechanism that is not fully established. Owing to the pivotal role of nuclear factor (NF)-κB in these responses, we tested the hypothesis that adenosine mediates its effects through suppression of NF-κB activation. We investigated the effects of adenosine on NF-κB activation induced by various inflammatory agents in human myeloid KBM-5 cells. The treatment of these cells with adenosine suppressed TNF-induced NF-κB activation, but had no effect on activation of another redox-sensitive transcription factor, AP-1. These effects were not restricted to myeloid cells, as NF-κB activation in other lymphocytic and epithelial cell types was also inhibited. The effect on TNF-induced NF-κB activation was selective as adenosine had minimal effect on NF-κB activation induced by H 2 O 2, PMA, LPS, okadaic acid, or ceramide, suggesting differences in the pathway leading to NF-κB activation by different agents. Adenosine also suppressed NF-κB-dependent reporter gene expression activated by TNF or by overexpression of TNFR1, TRAF 2, NIK, and p65 subunit of NF-κB. The suppression of TNF-induced NF-κB activation by adenosine was found not to be because of inhibition of TNF-induced IκBα phosphorylation and degradation or IκBα kinase activation. The suppression of TNF-induced NF-κB activation was unique to adenosine, as neither its metabolites (inosine, AMP, and ATP) nor pyrimidines (thymidine and uridine) had any effect. Overall, our results clearly demonstrate that adenosine selectively suppresses TNF-induced NF-κB activation, which may contribute to its role in suppression of inflammation and of the immune system.