Cyclic nucleotide levels within extending growth cones influence how navigating axons respond to guidance cues. Pharmacological alteration of cAMP or cGMP signalingin vitrodramatically modulates how growth cones respond to attractants and repellents, although how these second messengers function in the context of guidance cue signaling cascadesin vivois poorly understood. We report here that theDrosophilareceptor-type guanylyl cyclase Gyc76C is required for semaphorin-1a (Sema-1a)-plexin A repulsive axon guidance of motor axonsin vivo. Our genetic analyses define a neuronal requirement for Gyc76C in axonal repulsion. Additionally, we find that the integrity of the Gyc76C catalytic cyclase domain is critical for Gyc76C function in Sema-1a axon repulsion. Our results support a model in which cGMP production by Gyc76C facilitates Sema-1a-plexin A-mediated defasciculation of motor axons, allowing for the generation of neuromuscular connectivity in the developingDrosophilaembryo.