The p38 mitogen-activated protein (MAP) kinases are a family of serine/threonine protein kinases that play important roles in cellular responses to inflammation and external stress. Inhibitors of the p38 MAP kinase have shown promise for potential treatment of inflammatory disorders such as rheumatoid arthritis, acute coronary syndrome, psoriasis, and Crohn's disease. We identified a novel class of p38 inhibitors via high-throughput screening. PS200981 [3-(4-(1,4-diazepan-1-yl)-6-(((1S,2R,5S)-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)methylamino)-1,3,5-triazin-2-ylamino)-4-methylbenzamide], a representative compound identified from screening a collection of combinatorial libraries, amounting to 2.1 million compounds, inhibits p38alpha kinase and the lipopolysaccharide (LPS)-induced increase in tumor necrosis factor (TNF) alpha levels in cell media of human monocytes with IC50 values of 1 microM. The screening data revealed a preferred synthon, 3-amino-4-methyl benzamide, which is critical for the activity against p38. This synthon appeared almost exclusively in screening hits including PS200981, and slight variations of this synthon including 3-amino benzamide and 2-amino-4-methyl benzamide also contained in the library were inactive. PS200981 is equally potent against the alpha and beta forms of p38 but did not inhibit p38 gamma and is >25-fold selective versus a panel of other kinases. PS200981 inhibited the LPS-induced increase in TNFalpha levels when administered at 30 mg/kg to mice. Selectivity and in vivo activity of this class of p38 inhibitors was further demonstrated by PS166276 [(R)-3-(4-(isobutyl(methyl)-amino)-6-(pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)-4-methylbenzamide], a highly structurally related but more potent and less cytotoxic inhibitor, in several intracellular signaling assays, and in LPS-challenged mice. Overall, this novel class of p38 inhibitors is potent, active in vitro and in vivo, and is highly selective.