The induction of proliferation and differentiation in cultured mammalian cells is mediated by a cascade of protein phosphorylations. A key enzyme in this signaling pathway is mitogen-activated protein (MAP) kinase (or ERK, extracellular signal-regulated kinase). We report the recovery of a full-length cDNA clone encoding a MAP kinase from alfalfa. We have named the 44-kD protein encoded by this clone MsERK1. Recombinant MsERK1 (rMsERK1), when overexpressed in Escherichia coli, is recognized by antibodies raised against MAP kinases from rat, Xenopus, and sea star and by anti-phosphotyrosine antibodies. Site-directed mutagenesis of MsERK1 demonstrated that Tyr-215 is either directly or indirectly responsible for recognition of the protein by anti-phosphotyrosine antibodies. Semipurified rMsERK1 phosphorylated itself and a model substrate, myelin basic protein, in vitro, but the Tyr-215 mutant did neither. Genomic DNA gel blot analysis suggested that the gene that encodes MsERK1 is either a member of a small multigene family or a member of a polymorphic allelic series in alfalfa. Because MAP kinase activation has been associated with mitotic stimulation in animal systems, such an enzyme may play a role in the mitogenic induction of symbiotic root nodules on alfalfa by Rhizobium signal molecules.