Cyclin-dependent kinase 5 (Cdk5) in association with its neuronal activators p35 and p39 shows a complex involvement in the control of neurocytoskeletal dynamics. Here we show that alpha-chimaerin, a GTPase-activating protein specific for Rac and Cdc42, is a p35-binding protein. The interaction domains of p35 and alpha-chimaerin were delineated. In transfected HeLa cells, p35 and alpha-chimaerin displayed an overlapping distribution pattern and they could be co-immunoprecipitated from the cell lysate. As alpha-chimaerin has a regulatory function in actin repolymerization, these results suggested that the regulation of neurocytoskeleton dynamics by Cdk5 is mediated at least in part via alpha-chimaerin.