We examined the permeability of connexin36 (Cx36) homotypic gap junction (GJ) channels, expressed in neurons and β-cells of the pancreas, to dyes differing in molecular mass and net charge. Experiments were performed in HeLa cells stably expressing Cx36 tagged with EGFP by combining a dual whole-cell voltage clamp and fluorescence imaging. To assess the permeability of the single GJ channel (P(γ)), we used a dual-mode excitation of fluorescent dyes that allowed us to measure cell-to-cell dye transfer at levels not resolvable using whole-field excitation solely. We demonstrate that P(γ) of Cx36 for cationic dyes (EAM-1⁺ and EAM-2⁺) is ~10-fold higher than that for an anionic dye of the same net charge and similar molecular mass, Alexa fluor-350 (AFl-350⁻). In addition, P(γ) for Lucifer yellow (LY²⁻) is approximately fourfold smaller than that for AFl-350⁻, which suggests that the higher negativity of LY²⁻ significantly reduces permeability. The P(γ) of Cx36 for AFl-350 is approximately 358, 138, 23 and four times smaller than the P(γ)s of Cx43, Cx40, Cx45, and Cx57, respectively. In contrast, it is 6.5-fold higher than the P(γ) of mCx30.2, which exhibits a smaller single-channel conductance. Thus, Cx36 GJs are highly cation-selective and should exhibit relatively low permeability to numerous vital negatively charged metabolites and high permeability to K⁺, a major charge carrier in cell-cell communication.