||Neuronal Cdc2-like kinase is a heterodimer of a catalytic subunit of Cdk5 and a 25 kDa regulatory subunit that is derived from a 35 kDa protein. p35 is brain and neuronal specific and is designated neuronal Cdk5 activator, p35nck5a. An isoform of p35 has been found in human brain and this 39 kDa protein contains 367 amino acids and shares 57% amino acid sequence homology to p35. Like p35, p39 mRNA expression is also neuron specific and thus is designated neuronal Cdk5 activator isoform, p39nck5ai. Although the temporal and spatial distribution of p39i mRNA in mammals have been studied, the functional property of p39i protein has not been elucidated. A p39i specific polyclonal antibody has been produced in rabbit. This antibody detects a 42-kDa protein band in Western blot of whole rat brain lysate while it does not cross-react with p351/p25. Western blot analysis reveals that p39i protein is highly expressed at late embryonic stage in rat brain and the expression level is prominent till postnatal 1 week. During rat development, the expression level of p39i declines to minimal in adulthood. The antibody is also capable of immunoprecipitating baculovirus co-expressed Cdk5/p39i active complex and co-immunoprecipitating active Cdk5 from whole rat brain homogenates. Column chromatography techniques in combination with Western blotting and protein kinase assay analyses illustrate that p39i associates with and activates Cdk5 in rat brain. Previous study has reported that recombinant p39i protein can activate Cdk5 in vitro but whether p39i is the physiological activator of Cdk5 remains unclear. This report is the first to document the relationship between p39i and Cdk5 in vivo.