Coiled-coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization. Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve-muscle synapse, binds to the three-stranded coiled-coil domain of laminin-1. The interaction with laminin mediates the integration of agrin into basement membranes. Here we characterize the binding site within the laminin-1 coiled coil in detail. Binding assays with individual laminin-1 full-length chains and fragments revealed that agrin specifically interacts with the gamma1 subunit of laminin-1, whereas no binding to alpha1 and beta1 chains was detected. By using recombinant gamma1 chain fragments, we mapped the binding site to a sequence of 20 residues. Furthermore, we demonstrate that a coiled-coil conformation of this binding site is required for its interaction with agrin. The finding that recombinant gamma1 fragments bound at least 10-fold less than native laminin-1 indicates that the structure of the three-stranded coiled-coil domain of laminin is required for high-affinity agrin binding. Interestingly, no binding to a chimeric gamma2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.