We have isolated and characterized brain cDNA clones encoding microtubule-associated protein-2 (MAP-2) kinase for rat (rMNK1) and mouse (mMNK1). The nucleotide sequences diverged by only 5% whereas the amino acid sequences were identical except for one conservative residue change. Conservation of the expressed sequence extended into other mammalian species. These findings constitute the first demonstration of a strict evolutionary conservation of MAP-2 kinase. Genomic restriction patterns revealed a single MAP-2 kinase gene that shares homology with other genomic sequences. The 3' terminal half of the gene appears to be encoded by four exons. rMNK1 and mMNK1 differed from a recently reported MAP-2 kinase cDNA, termed ERK1, because of a nonconservative change in position 82, from Gly in ERK1 to Arg in rMNK1. The rMNK1 gene was found to be expressed mainly as a 1.8-kb transcript that was highest in brain and in lung. In contrast to ERK1, rMNK1 showed two equally prominent mRNA species in liver, at 1.8 kb and 5 kb, which imply differential processing of the primary transcript. Results derived from the immunological screening of an expression library showed that MAP-2 kinase might share epitopes with two prominent protein kinase C substrates, MARCKS (an 80-kD protein kinase C substrate) and GAP-43, suggesting the possibility that MAP-2 kinase could interact with kinase C.