Neuroligins 1−4 are postsynaptic transmembrane proteins capable of initiating presynaptic maturation via interactions with β-neurexin. Both neuroligins and β-neurexins have alternatively spliced inserts in their extracellular domains. Using analytical ultracentrifugation, we determined that the extracellular domains of the neuroligins sediment as dimers, whereas the extracellular domains of the β-neurexins appear monomeric. Sedimentation velocity experiments of titrated stoichiometry ratios of β-neurexin and neuroligin suggested a 2:2 complex formation. The recognition properties of individual neuroligins toward β-neurexin-1 (NX1β), along with the influence of their splice inserts, were explored by surface plasmon resonance and affinity chromatography. Different neuroligins display a range of NX1β affinities spanning more than 2 orders of magnitude. Whereas splice insert 4 in β-neurexin appears to act only as a modulator of the neuroligin/β-neurexin association, splice insert B in neuroligin-1 (NL1) is the key element regulating the NL1/NX1β binding. Our data indicate that gene selection, mRNA splicing, and post-translational modifications combine to give rise to a controlled neuroligin recognition code with a rank ordering of affinities for particular neurexins that is conserved for the neuroligins across mammalian species.