Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase (MAPK) have been used extensively in vitro and in vivo to investigate the role of p38 in physiological processes. As with other pharmacological inhibitors, non-specific targets of the p38 inhibitors have been reported. We have found that the protein kinase receptor interacting protein-2 (RIP2) is another target for the family of p38 inhibitors. The autophosphorylation of RIP2 was inhibited in vitro by the p38 inhibitors SB220025, SB203580 and PD169316 at concentrations comparable to those used to inhibit p38. We also identified two new in vitro substrates for RIP2, myelin basic protein and histone H3 with apparent Km values of 2.1 microM and 0.65 microM, respectively. The ability of RIP2 to phosphorylate these two substrates was sensitive to the p38 inhibitors as well. As was shown for p38alpha, a conserved threonine in the kinase domain of RIP2 is required for sensitivity to the inhibitors, indicating that the mechanism of inhibition of RIP2 is similar to that of p38. These results demonstrate that the pyridinyl imidazole inhibitors block RIP2 as well as p38 kinase activity.