The compound U0126 was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 also inhibited endogenous promoters containing AP-1 response elements but did not affect genes lacking AP-1 response elements. These effects were due to direct inhibition of the mitogen-activated protein kinase kinase family members MEK-1 and MEK-2. U0126 showed selective inhibition of MEK-1 and MEK-2, with little or no effect on other kinases. Kinetic analysis of U0126 and the MEK inhibitor PD098059 demonstrated that both compounds are noncompetitive inhibitors with respect to both MEK substrates, ATP, and ERK. U0126 and PD098059 bind to ΔN3-S218E/S222D MEK in a mutually exclusive manner, suggesting they may share a common or overlapping binding site. U0126 has approximately 100-fold higher affinity for ΔN3-S218E/S222D MEK than PD098059. U0126 also showed significant diminution in affinity for wild-type MEK compared to the mutant enzyme, indicating that the affinity is mediated by subtle conformational differences between the two activated forms of MEK. The selectivity, potency, and cellular efficacy of U0126 suggest that it will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.The compound U0126 was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 also inhibited endogenous promoters containing AP-1 response elements but did not affect genes lacking AP-1 response elements. These effects were due to direct inhibition of the mitogen-activated protein kinase kinase family members MEK-1 and MEK-2. U0126 showed selective inhibition of MEK-1 and MEK-2, with little or no effect on other kinases. Kinetic analysis of U0126 and the MEK inhibitor PD098059 demonstrated that both compounds are noncompetitive inhibitors with respect to both MEK substrates, ATP, and ERK. U0126 and PD098059 bind to ΔN3-S218E/S222D MEK in a mutually exclusive manner, suggesting they may share a common or overlapping binding site. U0126 has approximately 100-fold higher affinity for ΔN3-S218E/S222D MEK than PD098059. U0126 also showed significant diminution in affinity for wild-type MEK compared to the mutant enzyme, indicating that the affinity is mediated by subtle conformational differences between the two activated forms of MEK. The selectivity, potency, and cellular efficacy of U0126 suggest that it will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.