The basal lamina that ensheaths skeletal muscle fibers traverses the synaptic cleft at the neuromuscular junction. Synaptic and extrasynaptic portions of the basal lamina contain different laminin beta chains: beta 2 (or s) at synapses and beta 1 (or B1) extrasynaptically. Laminin beta 2 is also confined to synapselike patches on myotube surfaces in vitro, whereas beta 1 is present throughout the extracellular matrix. This differential localization of laminin beta chains was analyzed by expression of chimeric beta 1-beta 2 molecules in cultured mouse myotubes. A 16-amino acid carboxyl-terminal sequence in beta 2 was necessary for synaptic localization, and an amino-terminal domain in beta 1 promoted association with extracellular fibrils. The synaptic targeting sequence of beta 2 contains a site previously shown to be adhesive for motor neurons.