We report the purification to near homogeneity of a 45-kDa phorbol ester-stimulated protein kinase that phosphorylates and activates the Erk-1 gene product. This kinase, which we provisionally denote MEK for MAPK/Erk kinase, phosphorylated kinase-inactive Erk-1 protein primarily on a tyrosine residue and, to a lesser extent, on a threonine. We extend our previous results and show that two forms of purified MEK activated the myelin basic protein kinase encoded by Erk-1. MEK was inactivated by the serine/threonine phosphatase 2A but not by the protein-tyrosine phosphatase 1B. Sequence analysis of peptides generated by trypsin digestion of MEK revealed similarity to the proteins encoded by the Schizosaccharomyces pombe byr1 and Saccharomyces cerevisiae STE7 genes. These data are discussed with regard to a possible signal transduction mechanism.