Beta-dystrobrevin, a member of the dystrobrevin protein family, is a dystrophin-related and -associated protein restricted to non-muscle tissues and is highly expressed in kidney, liver and brain. Dystrobrevins are now thought to play an important role in intracellular signal transduction, in addition to providing a membrane scaffold in muscle, but the precise role of beta-dystrobrevin has not yet been determined. To study beta-dystrobrevin's function in brain, we used the yeast two-hybrid approach to look for interacting proteins. Four overlapping clones were identified that encoded Kif5A, a neuronal member of the Kif5 family of proteins that consists of the heavy chains of conventional kinesin. A direct interaction of beta-dystrobrevin with Kif5A was confirmed by in vitro and in vivo association assays. Co-immunoprecipitation with a monoclonal kinesin heavy chain antibody precipitated both alpha- and beta-dystrobrevin, indicating that this interaction is not restricted to the beta-dystrobrevin isoform. The site for Kif5A binding to beta-dystrobrevin was localized in a carboxyl-terminal region that seems to be important in heavy chain-mediated kinesin interactions and is highly homologous in all three Kif5 isoforms, Kif5A, Kif5B and Kif5C. Pull-down and immunofluorescence experiments also showed a direct interaction between beta-dystrobrevin and Kif5B. Our findings suggest a novel function for dystrobrevin as a motor protein receptor that might play a major role in the transport of components of the dystrophin-associated protein complex to specific sites in the cell.