Connexin-36 (Cx36) is the only gap junction protein that has been unambiguously identified in rodent pancreatic beta-cells. However, properties of gap junction channel unitary currents between beta-cells remain unrevealed. To address whether Cx36 forms functional channels in beta-cells, we characterized biophysical properties of macro- and microscopic junctional currents recorded from dual whole cell voltage clamp isolated pairs of dispersed mouse beta-cells. Electrical coupling was recorded in 80% of cell pairs with a junctional conductance (g(j)) of 355 +/- 45 pS (n = 20). Transjunctional voltage dependence was identified in three of seven cell pairs with high-input membrane resistances. Normalized steady-state g(j) (Gj) and transjunctional-voltage relation were well described by a two-state Boltzmann equation [maximal conductance (Gmax) = 1.0, voltage-insensitive conductance (Gmin) = 0.3 and 0.28, voltage gating sensitivity (A) = 0.21 and 0.23, and voltage at which one-half of the initial voltage-dependent conductance was reached (Vo) = -85 and 87 mV for negative and positive potentials, respectively]. Halothane reversibly uncoupled beta-cell pairs, and, during recovery, unitary conductances of 5-10 pS were recorded while using patch pipettes containing mainly CsCl. Although these properties are similar to those previously described for Cx36 channels in mammalian cell systems, we found that beta-cell junctional currents were insensitive to quinine. Cx36 transcript and protein expression in islets and freshly dispersed cell preparations was confirmed by RT-PCR and immunofluorescence. In conclusion, biophysical properties of junctional channels between beta-cells are similar but not identical to those previously described for homomeric Cx36 channels. Cell type-specific mechanisms that may account for these differences are discussed.