Mice in which netrin-G2 has been genetically inhibited do not startle to an acoustic stimulus, but otherwise perform normally through a behavioral test battery. Light microscopic examination of the inner ear showed no obvious structural abnormalities. Brainstem responses to acoustic stimuli (auditory brainstem responses, ABR) were also present, confirming the lack of any overarching defects in the inner ear or auditory nerve. Genetic inhibition of netrin-G2 ligand produced a nearly identical phenotype, that is, no startle with ABR present, and otherwise normal. This similarity confirms that these two proteins act in the same biological pathway. We have also determined that the affinity between the two proteins is strong, around 2.5 nM, similar to that observed between netrin-G1 and netrin-G1 ligand – 2.3 nM in our hands. The combination of equivalent phenotypes when genetically inhibited coupled with evidence of a strong biochemical interaction supports the notion of a receptor–ligand interaction between these two proteins in vivo. This interaction is critical for auditory synaptic responsiveness in the brain.