1. The dependence of gap junctional conductance on the intracellular concentrations of H+, Ca2+ and Mg2+ was studied in paired myocytes dissociated enzymatically from guinea-pig ventricle. To apply an internal solution buffered to specific H+, Ca2+ or Mg2+ concentration directly to one aspect of the gap junction, the non-junctional membrane of one of the pair was mechanically ruptured. The junctional conductance was measured by clamping the membrane potential of the other cell using a two-pipette voltage-clamp method. 2. The conductance of the non-junctional membrane was kept low in comparison with that of the junctional membrane (less than 1/50) by replacing both external and internal K+ with Cs+. 3. The current-voltage (I-V) relation of the junctional conductance was linear over the potential range examined (from -100 to +100 mV). No voltage or time dependence was detected. 4. The conductance of the gap junction between the paired cells ranged from 90 to 3900 nS with a peak distribution at 1000 nS. 5. The effect of H+ was examined over the pH range 7.4-5.4, while keeping the free-Ca2+ concentration at zero, or pCa 6.3 or 7.0 using 2-10 mM-EGTA. The junctional conductance was almost constant from pH 7.4 to 6.5 and decreased in a dose-dependent manner with further acidification. There was no difference in the pH-conductance relationships at various Ca2+ concentrations. The Hill coefficient was approximately 2.4 and the half-maximum concentration (pK'H) was 6.1. 6. The closing effect of Ca2+ on the gap junction channel was examined over the concentration range from pCa 7 to 5, while keeping the pH at 7.4, 7.0 or 6.5. At each pH, increasing Ca2+ decreased the junctional conductance with similar Hill coefficients of about 3.4. The pCa-conductance relationship shifted toward a higher Ca2+ concentration range as the pH was lowered (pK'Ca = 6.6, 6.4 and 5.6, at pH 7.4, 7.0 and 6.5, respectively). 7. Increasing Mg2+ also caused a fall in the junctional conductance over the pMg range 3.0-2.0 with a pK'Mg of 2.5 (3.2 mM), and a Hill coefficient of 3.0. 8. These results suggest that there are two respective binding sites for divalent cations and H+, and that the gap junctional conductance is regulated reversibly by the ligand-receptor reactions. Comparing the threshold concentrations of Ca2+ and H+ for electrical uncoupling, it was concluded that Ca2+ plays a more important role in regulating the gap junctional conductance of cardiac cells under physiological conditions.