The neuronal cyclin-dependent kinase p35/cdk5 comprises a catalytic subunit (cdk5) and an activator subunit (p35). To identify novel p35/cdk5 substrates, we utilized the yeast two-hybrid system to screen for human p35 binding partners. From one such screen, we identified β-catenin as an interacting protein. Confirmation that p35 binds to β-catenin was obtained by using glutathione S-transferase (GST)–β-catenin fusion proteins that interacted with both endogenous and transfected p35, and by showing that β-catenin was present in p35 immunoprecipitates. p35 and β-catenin also displayed overlapping subcellular distribution patterns in cells including neurons. Finally, we demonstrated that p35/cdk5 phosphorylates β-catenin. β-catenin also binds to presenilin-1 and altered β-catenin/presenilin-1 interactions may be mechanistic in Alzheimer's disease (AD). Abnormal p35/cdk5 activity has also been suggested to contribute to AD. We therefore investigated how modulation of p35/cdk5 activity influenced β-catenin/presenilin-1 interactions. Inhibition of p35/cdk5 with roscovitine did not alter the steady state levels of either β-catenin or presenilin-1 but reduced the amount of presenilin-1 bound to β-catenin. Thus, p35/cdk5 binds and phosphorylates β-catenin and regulates its binding to presenilin-1. The findings reported here therefore provide a novel molecular framework to connect p35/cdk5 with β-catenin and presenilin-1 in AD.