The Lutheran blood group glycoprotein (Lu), also known as basal cell adhesion molecule, is an Ig super-family transmembrane receptor for laminin α5. Lu is expressed on the surface of a subset of muscle and epithelial cells in diverse tissues and is thought to be involved in both normal and disease processes, including sickle cell disease and cancer. Here we investigated the binding of Lu to laminin α5 in vivo and in vitro. We prepared a soluble recombinant Lu (sol-Lu) composed of the Lu extracellular domain and a His6 tag. Sol-Lu bound specifically to laminin-10/11 (α5β1/β2γ1) in enzyme-linked immunosorbent assays and bound to bona fide basement membranes containing laminin α5 in tissue sections. Sol-Lu did not bind to tissue sections of laminin α5 knockout embryos, despite the fact that the four other a chains were present. To identify the Lu-binding site on laminin α5, we prepared modified α5 cDNAs encoding chimeric laminins containing all or part of the laminin α1 G domain in place of the analogous α5 regions. These constructs were used to generate transgenic mice. Proteins derived from transgenes were detected in basement membranes and were assayed for their ability to bind Lu by examining the localization of endogenous Lu and the binding of sol-Lu applied to tissue sections. Our results demonstrate that the α5 LG3 module is essential for Lu binding to laminin α5.