c-Src is phosphorylated at specific serine and threonine residues during mitosis in fibroblastic and epithelial cells. These sites are phosphorylated in vitro by the mitotic kinase Cdk 1 (p 341^cdc2). In contrast, c-Src in Y 79 human retinoblastoma cells, which are of neuronal origin, is phosphorylated at one of the mitotic sites, Ser 75, throughout the cell cycle. The identity of the serine kinase that nonmitotically phosphorylates c-Src on Ser 75 remains unknown. We now are able to show for the first time that Cdk 5 kinase, which has the same consensus sequence as the Cdk 1 and Cdk 2 kinases, is required for the phosphorylation in asynchronous Y 79 cells. The Ser 75 phosphorylation was inhibited in a dosedependent manner by butyrolactone I, a specific inhibitor of Cdk 5-type kinases. Three stable subclones that have almost no kinase activity were selected by transfection of an antisense Cdk 5-specific activator p 35 construct into Y 79 cells. The loss of the kinase activity caused an approximately 85% inhibition of the Ser 75 phosphorylation. These results present compelling evidence that Cdk 5/p 35 kinase is responsible for the novel phosphorylation of c-Sre at Ser 75 in neuronal cells, raising the intriguing possibility that c-Src acts as an effector of Cdk 5/p 35 kinase during neuronal development.