Four connexins (Cxs), mouse (m)Cx30.2, Cx40, Cx43, and Cx45, determine cell-cell electrical signaling in mouse heart, and Cx43 and Cx45 are known to form unapposed hemichannels. Here we show that mCx30.2, which is most abundantly expressed in sinoatrial and atrioventricular nodal regions of the heart, and its putative human ortholog, human (h)Cx31.9, also form functional hemichannels, which, like mCx30.2 cell-cell channels, are permeable to cationic dyes up to approximately 400 Da in size. DAPI uptake by HeLa cells expressing mCx30.2 was >10-fold faster than that by HeLa parental cells. In Ca(2+)-free medium, uptake of DAPI by HeLaCx30.2-EGFP cells was increased approximately 2-fold, but uptake by parental cells was not affected. Conversely, acidification by application of CO(2) reduced DAPI uptake by HeLaCx30.2-EGFP cells but had little effect on uptake by parental cells. Cells expressing mCx30.2 exhibited higher rates of DAPI uptake than did cells expressing any of the other cardiac Cxs. Cardiomyocytes of 2-day-old rats transfected with hCx31.9-EGFP took up DAPI and ethidium bromide 5-10 times faster than wild-type cardiomyocytes. Mefloquine, a close derivative of quinine and quinidine that exhibits antimalarial and antiarrhythmic properties, reduced conductance of cell-cell junctions and dye uptake through mCx30.2 hemichannels with approximately the same affinity (IC(50) = approximately 10 microM) and increased dependence of junctional conductance on transjunctional voltage. Unitary conductance of mCx30.2 hemichannels was approximately 20 pS, about twice the cell-cell channel conductance. Hemichannels formed of mCx30.2 and hCx31.9 may slow propagation of excitation in the sinoatrial and atrioventricular nodes by shortening the space constant and depolarizing the excitable membrane.