Mitogen-activated protein kinases, or extracellular signal-regulated kinases (ERKs), are serine/threonine protein kinases that are activated in response to a wide variety of extracellular stimuli and are encoded by a multigene family. Little is known about the function of the ERK-3 subfamily. To explore the molecular diversity of the ERK-3 subfamily, we isolated a novel human cDNA, designated Hu-ERK-3, from a fetal skeletal muscle library. Analysis of the complete 3,920-bp nucleotide sequence revealed that this clone encodes a predicted protein of 721 amino acids. In vitro transcription-translation generates a 97-kDa protein referred to as p97MAPK. Of all of the sequences compared, p97MAPK is the most homologous to rat ERK-3. Interestingly, although p97MAPK is highly (98%) homologous to ERK-3 at the amino acid level within the N-terminal two-thirds of the coding region, it diverges at the carboxyl terminus as a result of a unique extension of 178 amino acids. Although expression of p97MAPK was detected in all of the tissues tested by Northern (RNA) analysis, the most abundant expression was seen in skeletal muscle. An antibody raised against the unique C terminus recognized a 97-kDa protein in human cells. By using this antibody in an immune complex protein kinase assay, we have shown that treatment of human fibroblasts with serum or phorbol esters activates a myelin basic protein and histone H1 kinase activity in immunoprecipitates. p97MAPK appears to be the human homolog of rat ERK-3, and a member of this family is an active protein kinase.