In rodents, the vomeronasal system controls social and sexual behavior. However, several mechanistic aspects of sensory signaling in the vomeronasal organ remain unclear. Here, we investigate the biophysical basis of a recently proposed vomeronasal signal transduction component—a Ca2+-activated Cl− current. As the physiological role of such a current is a direct function of the Cl− equilibrium potential, we determined the intracellular Cl− concentration in dendritic knobs of vomeronasal neurons. Quantitative fluorescence lifetime imaging of a Cl−-sensitive dye at the apical surface of the intact vomeronasal neuroepithelium revealed increased cytosolic Cl− levels in dendritic knobs, a substantially lower Cl− concentration in vomeronasal sustentacular cells, and an apparent Cl− gradient in vomeronasal neurons along their dendritic apicobasal axis. Together, our data provide a biophysical basis for sensory signal amplification in vomeronasal neuron microvilli by opening Ca2+-activated Cl− channels.