During the formation of neuronal circuits, axon path-finding decisions specify the location of synapses on the correct brain side and in correct target areas. We investigated a possible link between axon midline crossing and the subsequent development of output synapses formed by these axons. Conditional knockout of Robo3 in the auditory system forced a large commissural synapse, the calyx of Held, to be exclusively formed on the wrong, ipsilateral side. Ipsilateral calyx of Held synapses showed strong transmission defects, with reduced and desynchron-ized transmitter release, fewer fast-releasable vesi-cles, and smaller and more variable presynaptic Ca 2+ currents. Transmission defects were not observed in a downstream inhibitory synapse, and some defects persisted into adulthood. These results suggest that axon midline crossing conditions functional maturation of commissural synapses, thereby minimizing the impact of mislocalized synap-ses on information processing. This mechanism might be relevant to human disease caused by mutations in the ROBO3 gene.