We have purified 3500-fold from rabbit skeletal muscle a 12,020-Da mitogen-activated protein kinase kinase (MEK)-enhancing factor (MEF) that stimulates both mitogen-activated protein kinase (MAPK) autophosphorylation and the rate (24-fold) at which the enzyme is phosphorylated by MEK in vitro. This was manifest by the finding that in the presence of MEF, molar equivalents of MEK to MAPK were sufficient to produce fully phosphorylated (2.1 +/- 0.4 mol/mol; S.D., n = 3) and activated MAPK. This contrasted with the 40:1 molar excess ratio of MEK to MAPK required to produce fully phosphorylated and activated MAPK in the absence of MEF. Phosphoamino acid analysis revealed that in the presence of MEF, phosphorylation of MAPK by MEK was ordered, with Tyr-185 phosphorylation preceding Thr-183 phosphorylation. However, the rate at which Thr-183 was phosphorylated relative to Tyr-185 was greatly increased. The finding that MEF stimulated MAPK autophosphorylation and increased its ability to be phosphorylated by MEK suggests a mechanism of action in which MEF interacts with MAPK to alter its conformation.