Mitogen-activated protein kinase (MAPK) activation is an important signal involved in regulating cellular proliferation and/or differentiation. The immediate upstream kinase MAPK kinase, referred to as MEK, activates MAPK by phosphorylation on specific tyrosine and threonine residues. To date, two MEK's have been cloned and characterized, MEK1 and MEK2. Here we report the cloning of MEK2b from mouse pituitary cDNA. Rat and mouse MEK2 amino acid sequences vary by only three amino acids; the three changes are conserved in the MEK1 sequence. Analysis of recombinant MEK2b and MEK1 demonstrated similar activation by upstream kinases and phosphotransferase activity toward MAPK, while they differed in autophosphorylation and the ability to serve as a substrate for MAPK. The findings demonstrate significant differences in potential regulatory mechanisms of MEK1 and MEK2/2b but not in their activation by upstream regulators.