Neuroligins, proteins of the alpha/beta-hydrolase fold family, are found as postsynaptic transmembrane proteins whose extracellular domain associates with presynaptic partners, proteins of the neurexin family. To characterize the molecular basis of neuroligin interaction with neurexin-beta, we expressed five soluble and exportable forms of neuroligin-1 from recombinant DNA sources, by truncating the protein before the transmembrane span near its carboxyl terminus. The extracellular domain of functional neuroligin-1 associates as a dimer when analyzed by sedimentation equilibrium. By surface plasmon resonance, we established that soluble neuroligins-1 bind neurexin-1beta, but the homologous alpha/beta-hydrolase fold protein, acetylcholinesterase, failed to associate with the neurexins. Neuroligin-1 has a unique N-linked glycosylation pattern in the neuroligin family, and glycosylation and its processing modify neuroligin activity. Incomplete processing of the protein and enzymatic removal of the oligosaccharides chain or the terminal sialic acids from neuroligin-1 enhance its activity, whereas deglycosylation of neurexin-1beta did not alter its association capacity. In particular, the N-linked glycosylation at position 303 appears to be a major determinant in modifying the association with neurexin-1beta. We show here that glycosylation processing of neuroligin, in addition to mRNA splicing and gene selection, contributes to the specificity of the neurexin-beta/neuroligin-1 association.