Mode I phosphorylated MAP1B is observed in developing and pathogenic brains. Although Cdk5 has been believed to phosphorylate MAP1B in the developing cerebral cortex, we show that a Cdk5 inhibitor does not suppress mode I phosphorylation of MAP1B in primary and slice cultures, while a JNK inhibitor does. Coincidently, an increase in phosphorylated MAP1B was not observed in COS7 cells when Cdk5 was cotransfected with p35, but this did occur with p25 which is specifically produced in pathogenic brains. Our primary culture studies showed an involvement of Cdk5 in regulating microtubule dynamics without affecting MAP1B phosphorylation status. The importance of regulating microtubule dynamics in neuronal migration was also demonstrated by in utero electroporation experiments. These findings suggest that mode I phosphorylation of MAP1B is facilitated by JNK but not Cdk5/p35 in the developing cerebral cortex and by Cdk5/p25 in pathogenic brains, contributing to various biological events.