MAPK-p38 plays an important role in inflammation. Several studies have shown that blocking p38 activity attenuates the transcriptional activity of the proinflammatory transcription factor NF-kappaB without altering its DNA-binding activity. We have also observed that blocking p38 in human primary astrocytes suppresses the transcriptional but not the DNA-binding activity of NF-kappaB and down-regulates the expression of an NF-kappaB-dependent gene, inducible NO synthase. However, the molecular mechanism of p38-mediated regulation of NF-kappaB remains largely unknown. In this study, we delineate that p38 controls the transcriptional activity of NF-kappaB by regulating acetylation of p65, but not its phosphorylation. The combination of IL-1beta and IFN-gamma, previously shown to strongly induce inducible NO synthase in human primary astrocytes, induced p38-dependent phosphorylation of acetyltransferase coactivator p300, but not p65, and subsequent association of p300 with p65. Furthermore, immunocomplex-histone acetyltransferase assays demonstrated that cytokine-induced association of p65 with biologically active immunocomplex-histone acetyltransferase assay was dependent on p38. It has been previously reported that acetylation of p65 at K310 residue is important for transcriptional activity of NF-kappaB. Accordingly, we found that cytokine-induced association of p65 with p300 led to acetylation of p65 at K310. Because p38 regulated the association between p65 and p300, blocking p38 activity also led to attenuation of p65-K310 acetylation in cytokine-stimulated astrocytes. Taken together, this study illuminates a novel regulatory role of p38 during neuroinflammation where this MAP kinase controls acetylation of NF-kappaB p65 by regulating acetyltransferase activity of coactivator p300.