Partial exposure of single ventricular myocytes to membrane-permeant weak acids or bases, using a dualmicroperfusion technique, generates large and stable intracellular pH (pH_i) gradients. In this paper, we have investigated the feasibility of using the technique to estimate junctional proton permeability. This was done by recording the pH_i-gradient developed across the junctional region of a pair of conjoined ventricular myocytes, isolated enzymically from guinea-pig heart, when one of the cells was partially exposed to acetate or ammonium. We show that under Hepes-buffered conditions, the junctional discontinuity in the pH_i-profile can be used to derive an apparent proton permeability coefficient (P_H^app). The mean P_H^app obtained was 4.45±0.21^.10^-4 cm/s (N=43) at an average junctional pH_i of 7.04±0.02. In the presence of the junctional inhibitor, -glycyrrhetinic acid, exposure of the proximal cell to weak acid or base produced no pH_i change in the distal cell, confirming that distal changes were normally caused by acid/base flux through connexons assembled into junctional channels. The validity of the dualmicroperfusion method was tested further by using a diffusion-permeation-reaction model for intracellular protons, designed to highlight possible errors in the estimates of P_H ^app. Our present technique for measuring P_H^app provides a useful alternative to the previous, more invasive technique of locally loading acid through a cell-attached patch-pipette. The technique may provide a simple method for investigating the factors regulating cell-to-cell proton transmission.