Lens fiber cells contain two gap junction proteins (Cx56 and Cx45.6 in the chicken). Biochemical studies have suggested that these two proteins can form heteromeric connexons. To investigate the biophysical properties of heteromeric lens connexons, Cx56 was co-expressed with Cx45.6 (or its mouse counterpart, Cx50) in Xenopus oocytes. Whole-cell and single-channel currents were measured in single oocytes by conventional two-microelectrode voltage-clamp and patch clamp techniques, respectively. Injection of Cx56 cRNA induced a slowly activating, nonselective cation current that activated on depolarization to potentials higher than -10 mV. In contrast, little or no hemichannel current was induced by injection of Cx50 or Cx45.6 cRNA. Co-expression of Cx56 with Cx45.6 or Cx50 led to a shift in the threshold for activation to -40 or -70 mV, respectively. It also slowed the rate of deactivation of the hemichannel currents. Moreover, an increase in the unitary conductance, steady state probability of hemichannel opening and mean open times at negative potentials, was observed in (Cx56 + Cx45.6) cRNA-injected oocytes compared with Cx56 cRNA-injected oocytes. These results indicate that co-expression of lens fiber connexins gives rise to novel channels that may be explained by the formation of heteromeric hemichannels that contain both connexins.