The organ of Corti has been found to have multiple gap junction subunits, connexins, which are localized solely in nonsensory supporting cells. Connexin mutations can induce sensorineural deafness. However, the characteristics and functions of inner ear gap junctions are not well known. In the present study, the voltage-dependence of gap junctional conductance (G(j)) in cochlear supporting cells was examined by the double voltage clamp technique. Multiple types of asymmetric voltage dependencies were found for both nonjunctional membrane voltage (V(m)) and transjunctional (V(j)) voltage. Responses for each type of voltage dependence were categorized into four groups. The first two groups showed rectification that was polarity dependent. The third group exhibited rectification with either voltage polarity, i.e., these cells possessed a bell-shaped G(j)-V(j) or G(j)-V(m) function. The rectification due to V(j) had fast and slow components. On the other hand, V(m)-dependent gating was fast (<5 msec), but stable. Finally, a group was found that evidenced no voltage dependence, although the absence of V(j) dependence did not preclude V(m) dependence and vice versa. In fact, for all groups V(j) sensitivity could be independent of V(m) sensitivity. The data show that most gap junctional channels in the inner ear have asymmetric voltage gating, which is indicative of heterogeneous coupling and may result from heterotypic channels or possibly heteromeric configurations. This heterogeneous coupling implies that single connexin gene mutations may affect the normal physiological function of gap junctions that are not limited to homotypic configurations.