Local perfusion of one end of an isolated ventricular myocyte with membrane-permeant weak acids or bases results in a large and stable gradient of intracellular pH as measured using confocal imaging of carboxy-SNARF-1 (Spitzer et al. 2000). This pH_i gradient occurs because intracellular H⁺ mobility is low compared with that of the weak acid/base. We have adapted this technique to both measure and model H⁺ permeation across the myocardial gap junction. The proximal cell of an enzymically isolated end-to-end ventricular myocyte pair (from humanely killed guinea pig hearts) was partially perfused with Hepes-buffered Tyrode solution containing NH₄Cl (20-30mM) or Na-acetate (80-120mM). A pH_i gradient was generated down the length of the cell-pair, with a step change of pH_i occurring across the junctional region. The junctional inhibitor, α-glycyrrhetinic acid (60μM), prevented any pHi change from occurring in the distal cell (n=25), while the Na⁺-H⁺ exchange inhibitor, cariporide (30μM), exerted no effect, indicating that acid normally translocates the gap junction through connexin channels (Zaniboni et al. 2003). Pre- and post-junctional slopes of the pHi profile were used in conjunction with the apparent H⁺ diffusion coefficient to estimate junctional acid flux, driven passively by the junctional pH_i gradient. Modelling of these parameters yielded an estimate of the apparent junctional proton permeability constant (P_H^app). This was converted into a mobile buffer permeability constant (P_mob) on the assumption that protons permeate the junction when conjugated to mobile buffers (Ref 2). At a physiological resting junctional pH of 7.04±0.02, P_mob was found to be 21.3±1.0×10^-4 cm/s (n=43). P_mob was also measured after cell-pairs had been acid-loaded (by uniformly prepulsing with 30mM NH₄Cl before applying the dual microstream). At a mean junctional pH_i of 6.57±0.05, P_mob was 14.2±1.6×10^-4 cm/s (n=19), a reduction of 33%, showing that a significant intracellular acidosis reduces cell-to-cell proton transmission. When cell-pairs were loaded with 100μM BAPTA-AM for 10 min, P_mob was reduced from 20.0±1.6×10^-4 cm/s (n=37) at a junctional pH_i of 7.02±0.02, to 16.8±2.1×10^-4 cm/s (n=25) at a junctional pHi of 6.59±0.04. Although this represents a 16% reduction, the difference was not significant (P>0.05). This suggests that over the pH_i range tested, some of the acid-inhibition of proton transmission between cells is dependent on possible changes in [Ca²⁺]_i.