We have identified and purified from bovine brain a novel protein kinase which catalyzes in vitro phosphorylation of neurofilament proteins NF-H and NF-M and tau proteins at sites implicating the enzyme in the regulation of neurocytoskeleton dynamics and in Alzheimer pathology. The protein kinase displays a phosphorylation site specificity similar or identical to the cell cycle regulatory kinase, cdc2 kinase. The purified kinase is a heterodimer of a cdc2-like catalytic subunit, called cdk5, and a 25 kDa regulatory subunit. The regulatory subunit is essential for kinase activity, and it is derived from a 35 kDa protein, p35 by proteolysis. Northern blot analysis of tissue distribution indicates that cdk5 is widely distributed but especially rich in brain, whereas p35 expression is only found in brain. The protein kinase is therefore termed neuronal cdc2-like kinase. The neuron-specificity of the enzyme appears to be conferred by the regulatory subunit. During cell division, cdc2 kinase is regulated by complex phosphorylation mechanisms involving a network of specific protein kinases. Some of these kinases or their homologs have been found in mammalian brains and they may be involved in the regulation of neuronal cdc2-like kinase.