The c-Kit receptor protein-tyrosine kinase is tightly regulated in normal cells, whereas deregulated c-Kit kinase activity is implicated in the pathogenesis of human cancers. Crystal structures of c-Kit kinase reveal a manner in which the small juxtamembrane domain functions to maintain the kinase in an autoinhibited state. Insertion of this autoinhibitory domain into the cleft between the kinase N- and C-lobes flips the DFG motif into its off state, thereby inducing the activation loop to fold back over from its extended conformation in the active kinase where it binds as a pseudosubstrate at the kinase-active center.