The cytoskeletal matrix assembled at active zones (CAZ) is implicated in defining neurotransmitter release sites. However, little is known about the molecular mechanisms by which the CAZ is organized. Here we report a novel interaction between Piccolo, a core component of the CAZ, and GIT proteins, multidomain signaling integrators with GTPase-activating protein activity for ARF small GTPases. A small region (∼150 aa residues) in Piccolo, which is not conserved in the closely related CAZ protein Bassoon, mediates a direct interaction with the SHD domain of GIT1. Piccolo and GIT1 colocalize at synaptic sites in cultured neurons. In brain, Piccolo forms a complex with GIT1 and various GIT-associated proteins including bPIX, focal adhesion kinase, liprin-a and paxillin. Point mutations in the SHD of GIT1 differentially interfere with the association of GIT1 with Piccolo, bPIX and focal adhesion kinase, suggesting that these proteins bind to the SHD by different mechanisms. Intriguingly, GIT proteins form homo- and heteromultimers through their C-terminal GRKBD domain in a tail-to-tail fashion. This multimerization enables GIT1 to simultaneously interact with multiple SHD-binding proteins including Piccolo and bPIX. These results suggest that, through their multimerization and interaction with Piccolo, the GIT family proteins are involved in the organization of the CAZ.