A subset of connexins can form unopposed hemichannels in expression systems providing an opportunity to compare hemichannel gating properties with those of intact gap junction channels. Zebrafish Cx35 is a member of the Cx35/Cx36 subgroup of connexins highly expressed in the retina and brain. Here we show that Cx35 expression in Xenopus oocytes and N2A cells produced large outward whole cell currents on cell depolarization. Using whole-cell, cell-attached and excised patch configurations we obtained multichannel and single channel current recordings attributable to the Cx35 hemichannels (I_hc) that were activated and increased by stepwise depolarization of V_m and deactivated by hyperpolarization. The currents were not detected in untransfected N2A cells, or control oocytes injected with antisense Cx38. However, water injected oocytes that were not antisense treated showed activities attributable to Cx38 hemichannels that were easily distinguishable from Cx35 hemichannels by a significantly larger unitary conductance (250-320 pS). The unitary conductance (_hc) of Cx35 hemichannels exhibited a pronounced V_m-dependence i.e. _hc increased/decreased with relative hyperpolarization/depolarization (_hc was 72 pS at V_m=-100 mV and 35 pS at V_m=100 mV). Extrapolation to V_m=0 mV predicted a _hc of 48 pS, suggesting a unitary conductance of intact Cx35 gap junction channels of ~24 pS. Channel gating was also V_m-dependent, open time declined with negative V_m, and increased with positive V_m. The ability to break down the complex gating of intact intercellular channels into component hemichannels in vitro will help to evaluate putative physiological roles for hemichannels in vivo.