Neuronal Cdc2-like kinase is a heterodimer of Cdk5 and a 25-kDa subunit that is derived from a 35-kDa brain- and neuron-specific protein called the neuronal Cdk5 activator (p35/p25nck5a) (Lew, J., Huang, Q.-Q., Qi, Z., Winkfein, R. J., Aebersold, R., Hunt, T., and Wang, J. H. (1994) Nature 371, 423-426; Tsai, L. H., Delalle, I., Caviness, V. S., Jr., Chae, T., and Harlow, E. (1994) Nature 371, 419-423). Upon screening of a human hippocampus library with a bovien Nck5a cDNA, we uncovered a distinct clone encoding a 39-kDa isoform of Nck5a. The isoform, designated the neuronal Cdk5 activator isoform (p39nck5ai), showed a high degree of sequence similarity to p35nck5a with 57% amino acid identity. Northern blot analysis detected its mRNA transcript in bovine and rat cerebrum and cerebellum, but not in any other rat tissues examined. In situ hybridization showed that Nck5ai was enriched in CA1 to CA3 of the hippocampus, but absent in the fimbria of hippocampal formation. Among seven cell lines in proliferating cultures, only PC12 and N2A, two cell lines capable of differentiating into neuron-like cells, were found to contain Nck5ai mRNA. A 30-kDa truncated form of Nck5ai expressed as a glutathione S-transferase fusion protein in Escherichia coli] was found to associate with Cdk5 to form an active Cdk5 kinase. Thus, the isoform shares many common characteristics with p35nck5a, including Ckd5 activating activity and brain- and neuron-specific expression. Both proteins show limited sequence homology to cyclins, suggesting that they define a new family of cyclin-dependent kinase-activating proteins.