Extracellular signal-regulated kinase 7 (ERK7) shares significant sequence homology with other members of the ERK family of signal transduction proteins, including the signature TEY activation motif. However, ERK7 has several distinguishing characteristics. Unlike other ERKs, ERK7 has been shown to have significant constitutive activity in serum-starved cells, which is not increased further by extracellular stimuli that typically activate other members of the mitogen-activated protein kinase (MAPK) family. On the other hand, ERK7's activation state and kinase activity appear to be regulated by its ability to utilize ATP and the presence of its extended C-terminal region. In this study, we investigated the mechanism of ERK7 activation. The results suggest that 1) MAPK kinase (MEK) inhibitors do not suppress ERK7 kinase activity; 2) intramolecular autophosphorylation is sufficient for activation of ERK7 in the absence of an upstream MEK; and 3) multiple regions of the C-terminal domain of ERK7 regulate its kinase activity. Taken together, these results indicate that autophosphorylation is sufficient for ERK7 activation and that the C-terminal domain regulates its kinase activity through multiple interactions.