The ERK3 cDNA predicts a protein of 62,000 in size with a C-terminal domain that extends 180 amino acids beyond the conserved core of ERK family protein kinases. Immunoblotting with antibodies raised to recombinant protein and to peptides from the catalytic core and three regions of the C-terminal tail revealed that ERK3 is the expected size and is ubiquitously expressed in a variety of cell lines and tissues. ERK3, unlike the MAP kinases ERK1 and ERK2, is localized in the nucleus in exponentially growing, quiescent, and growth factor-stimulated cells. If the 180 amino acids at its C terminus are deleted, the resulting ERK3 fragment of 45 kDa is still found primarily in the nucleus, indicating that the C terminus is not required for its localization. Recombinant ERK3 expressed in mammalian cells or in bacteria is a protein kinase, as deduced from its capacity to autophosphorylate. Mutation of a conserved residue (Asp171) expected to be involved in catalysis eliminated autophosphorylation. Ser189 of ERK3, which corresponds to Thr183, one of the activating phosphorylation sites of ERK2, is autophosphorylated in vitro and phosphorylated in vivo. Despite marked similarities to ERK1 and ERK2, ERK3 does not phosphorylate typical MAP kinase substrates, indicating that it has distinct functions.