The atypical cyclin-dependent kinase 5 (Cdk5) serves an array of different functions in cell biology. Among these are axonal guidance, regulation of intercellular contacts, cell differentiation, and prosurvival signaling. The variance of these functions suggests that Cdk5 activation comes to pass in different cellular compartments. The kinase activity, half-life, and substrate specificity of Cdk5 largely depend on specific activators, such as p25, p35, p39, and cyclin I. We hypothesized that the subcellular distribution of Cdk5 activators also determines the localization of the Cdk5 protein and sets the stage for targeted kinase activity within distinct cellular compartments to suit the varying roles of Cdk5. Cdk5 localization was analyzed in murine kidney and brain slices of wild-type and cyclin I- and/or p35-null mice by immunohistochemistry and in cultured mouse podocytes using immunofluorescence labeling, as well as cell fractionation experiments. The predominance of cyclin I mediates the nuclear localization of Cdk5, whereas the predominance of p35 results in a membranous localization of Cdk5. These findings were further substantiated by overexpression of cyclin I and p35 with altered targeting characteristics in human embryonic kidney 293T cells. These studies reveal that the subcellular localization of Cdk5 is determined by its specific activators. This results in the directed Cdk5 kinase activity in specific cellular compartments dependent on the activator present and allows Cdk5 to serve multiple independent roles.