The inclusion of 1mM of the membrane-permeant aldehyde, 2-nitrobenzaldehyde (NBA) in superfusion solutions has no major side-effects on rat ventricular myocyte contractility or intracellular pH (pH_i) but does provide substrate for photolytic release of protons by flashes of UV laser light. Myocytes were enzymically isolated from the ventricles of humanely killed rats. Cells and cell-pairs were AM-loaded with the fluorophore carboxy-SNARF-1 or carboxy-SNARF-4F for confocal imaging of pH_i. Whole-cell exposure to a flash of UV light (pixel dwell time 9.8μs) produced a uniform fall of pH_i of ~0.01 pH_i units, equivalent to an acid load of 0.22±0.07 mM (n=7). The release of acid can be confined to a region of interest (as small as 4 by 4μm) to investigate H⁺_i mobility by following the relaxation of pH_i after a burst (1Hz for 6s) of UV flashes or by following the progress of acid-loading between consecutive UV flashes (0.1Hz for 60s). Since flash photolysis produces large acid-loads, it can also be adapted to measure junctional transmission of acid between coupled cells. Under Hepes-buffered conditions and in the presence of 30μM cariporide to block sarcolemmal Na⁺-H⁺ transport, the apparent proton diffusion coefficient (D_H^app) at resting pH_i determined by each photolytic method was 13.4±1.63×10^-7 cm²/s (N=46, burst of flashes) and 12.2±1.04×10^-7 cm²/s (N=29, continuous flashing), respectively. These were similar to the previously determined value of D_H^app using pipette-loading of acid (Zaniboni et al, 2003, Am J Physiol 285, H1236-46). By manipulating starting pH_i (by means of a whole-cell ammonium or acetate prepulse), D_H^app was shown to rise four-fold over the pH_i range 6.4 to 7.4. The apparent junctional permeability constant for protons (P_H^app) was 1.9±0.5×10^-5 cm/s (N=17) for side-by-side pairs at a mean pH_i of 7.13±0.04. Photolytic uncaging of protons offers a new and less invasive method of studying proton mobility than by using pipette acid-loading. This technique can also potentially be used to study H⁺_i mobility in intact myocardium and even perfused hearts. This work was supported by a British Heart Foundation Programme Grant (RDVJ).