The adult mammalian cortex is characterized by a distinct laminar structure generated through a well-defined pattern of neuronal migration. Successively generated neurons are layered in an "inside-out" manner to produce six cortical laminae. We demonstrate here that p35, the neuronal-specific activator of cyclin-dependent kinase 5, plays a key role in proper neuronal migration. Mice lacking p35, and thus p35/cdk5 kinase activity, display severe cortical lamination defects and suffer from sporadic adult lethality and seizures. Histological examination reveals that the mutant mice lack the characteristic laminated structure of the cortex. Neuronal birth-dating experiments indicate a reversed packing order of cortical neurons such that earlier born neurons reside in superficial layers and later generated neurons occupy deep layers. The phenotype of p35 mutant mice thus demonstrates that the formation of cortical laminar structure depends on the action of the p35/cdk5 kinase.