Gap junctions are thought to mediate the direct intercellular coupling of adjacent cells by the gating of an aqueous pore permeable to ions and molecules of up to 1 kD or 8 to 14 Å in diameter. We performed ion-substitution and dye-transfer experiments to determine the relative Cl−/K+conductance and dye permeability of anionic fluorescein derivatives in chick connexin45 (Cx45) channels. We demonstrate that Cx45 forms a 26±6-picosiemen (pS) channel with a maximum detectable Cl−permeability of 0.2 relative to K+or Cs+. Although homogeneous channel conductances were observed in multichannel recordings, the open probability estimates were indicative of nonhomogeneous gating behavior and occasional cooperativity. A second conductance state of 19±4 pS begins to predominate at higher voltages. Cx45 gap junctions are permeable to 2′,7′-dichlorofluorescein but are not permeable to the more polar 6-carboxyfluorescein dye. These observations suggest that the Cx45 pore diameter is ≈ 10 Å and is associated with a fixed negative charge within the junctional channel.