Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X7. In this regard, following exposure to bacterial lipopolysaccharide (LPS), P2X7 activation augments the expression of the inducible nitric oxide synthase and the production of nitric oxide in macrophages. Because P2X7 has been reported to stimulate certain members of the MAP kinase family (ERK-1/ERK-2) and can enhance the DNA-binding activity of nuclear factor-B (NF-B), we tested the hypothesis that LPS and nucleotides regulate NF-B-dependent inflammatory events via crosstalk with MAP kinase-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X7 selective ligand 3'-O-(4 benzoyl benzoic) adenosine 5'-triphosphate (BzATP) results in the cooperative activation of NF-B DNA binding activity and a sustained attenuation of the levels of the NF-B inhibitory protein, IB. Interestingly, a persistent reduction in IB levels is also observed when the MEK1/2 inhibitor, U0126, is co-administered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating IB protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in IB levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage co-treatment with BzATP and LPS when compared to the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-B/IB-dependent inflammatory mediator production, and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-B and Ras/MEK/MAP kinase associated-pathways.