The membrane-permeant caged-H⁺ substance, 2-nitrobenzaldehyde (NBA), undergoes intramolecular redox catalysed by UV light. The product of photolysis is a strong acid, 2-nitrosobenzoic acid (pK=2.2), which is trapped intracellularly and which releases H⁺. Rat ventricular myocytes, AM-loaded with the pH-sensitive dye, carboxy-SNARF-1, were superfused (37°C) with Hepes-buffered Tyrode solution containing 1mM NBA and 30μM cariporide (to inhibit Na⁺-H⁺ exchange). In the absence of UV light, NBA itself had no effect on resting pH, cell contraction or evoked Ca²⁺ transients. Myocytes were imaged confocally (Leica TCS NT) while excitation was provided alternately by an Argon laser (514nm) to image intracellular pH (pH_i) and by a UV laser (351nm, 20mW) to flash-photolyse NBA. At 100% UV laser power, the quantum yield of NBA photolysis reaches 65% (pixel dwell time <10µs). The magnitude and area of H⁺ release can be controlled through neutral density filters and by limiting the scanning window, respectively. Exposing a small region (typically 4×4µm) of a cell to repetitive (0.11Hz) flashes of UV light, interrupted by a period of pH_i data acquisition, produces a local cumulative rise in [H⁺] that dissipates by diffusion. The time-course of [H⁺] rise in selected regions of a single myocyte can be used to measure the apparent H⁺ diffusion coefficient (D_H^app), estimated as 13.4±1.6×10^-7 cm²/s (n=46) using diffusion equations. This technique was also applied to cell-pairs to measure the apparent junctional H⁺ permeability constant (P_H^app). The time-course of [H⁺] rise in side-by-side and end-to-end myocyte pairs gave an estimate for P_H^app of 0.35±0.05×10^-4 cm/s (n=21) and 3.6±1.1×10^-4 cm/s (n=7), respectively (significant anisotropy of permeation). Replacing Hepes with 5% CO₂/bicarbonate in the superfusion solutions increases D_H^app and P_H^app by ~30-40%. This is consistent with the role of CO₂/bicarbonate, an open buffer, in facilitating intracellular H⁺ mobility. The present technique may also be useful for studying the spread of acidosis within multicellular cardiac preparations, including localised regions of the whole-heart, and in studying the spatial interactions between pH and other signalling molecules such as Ca²⁺.