The multidomain shank/ProSAP/SSTRIP proteins are major scaffold proteins in glutamatergic synapses in the mammalian brain; expression of shank1/SSTRIP in hippocampal neurons induces morphological changes in dendritic spines, suggesting that shank1 is involved in synapse formation and activity-dependent changes of synaptic structure. Using part of the proline-rich region of shank1 in a yeast two hybrid screen, we identified the insulin receptor substrate IRSp53 as an interaction partner. Overlay assays verified a strong interaction between a proline-rich sequence (residues 911-940) in shank1 and the SH3 domain of IRSp53. When coexpressed in HEK cells, shank1 colocalizes with IRSp53 in intracellular structures, preventing targeting of IRSp53 to filopodia which are induced by IRSp53 expression in the absence of shank1. IRSp53 also binds to the activated form of the small G-protein cdc42. Interestingly, IRSp53 coprecipitates with shank1 from transfected HEK cells in a small G-protein-regulated manner. Thus, IRSp53 constitutes a cdc42-regulated ligand for shank1 which may provide a molecular basis for small G-protein mediated effects on the structure of the postsynaptic complex.