AbstractTargeting cytokines has become an important focus in the treatment of many inflammatory disorders. p38 MAP kinase (MAPK) is the key enzyme in regulating the biosynthesis and release of pro‐inflammatory cytokines such as IL‐1β and TNFα. Inhibition of p38 MAPK results in decreased expression of these cytokines. Tri‐ and tetrasubstituted pyridinylimidazoles are potent inhibitors of p38 MAPK. Substitution on the pyridinyl moiety allows the design of inhibitors that show increased selectivity and activity by targeting the enzyme's hydrophobic region II. The objective of this study was to synthesize novel 1,2,4,5‐tetrasubstituted imidazole derivates and to characterize them not only for their ability to inhibit p38 MAPK and modulate cytokine release in human whole blood, but also to evaluate their metabolic stability. Biological data and metabolic studies demonstrate that the introduction of a 2‐acylamino function at C2 of the pyridine results in highly efficient and metabolically stable inhibitors relative to C2‐alkylamino derivatives. A series of novel candidates was investigated for metabolic stability in human liver microsomes and in human whole blood. Additionally, metabolic S‐oxidation was investigated, and possible metabolites were synthesized.