The collagen-tailed form of acetylcholinesterase (AChE) is concentrated at the vertebrate neuromuscular junction (NMJ), where it is responsible for rapidly terminating neurotransmission. This unique oligomeric form of AChE, consisting of three tetramers covalently attached to a collagen-like tail, is more highly expressed in innervated regions of skeletal muscle fibers, where it is externalized and attached to the synaptic basal lamina interposed between the nerve terminal and the receptor-rich postsynaptic membrane. Although it is clear that the enzyme is preferentially synthesized in regions of muscle contacted by the motoneuron, the molecular events underlying its localization to the NMJ are not known. Here we show that perlecan, a multifunctional heparan sulfate proteoglycan concentrated at the NMJ, is the unique acceptor molecule for collagen-tailed AChE at sites of nerve-muscle contact and is the principal mechanism for localizing AChE to the synaptic basal lamina.