A rapid fluorescence imaging system was developed and utilised to investigate the time-course of intracellular calcium concentration ([Ca2+]i) gradients generated by action potentials in CA1-CA3 pyramidal cells within brain slices of the rat hippocampus. The system, which is based on a fast commercial CCD camera, can acquire hundreds of 128 x 128 pixel images in sequence, with minimal inter-frame interval of 2.5 ms (400 frames/s) and 12 bit/pixel accuracy. By synchronising patch clamp recordings with image capture, the timing of transmembrane potential variation, ionic Ca2+ current and Ca2+ diffusion were resolved at the limit of the relaxation time for the dye-Ca2+ binding reaction (approximately 5 ms at room temperature). Numerical simulations were used to relate measured fluorescence transients to the spatio-temporal distribution of intracellular Ca2+ gradients. The results obtained indicate that dye reaction-diffusion contributes critically to shaping intracellular ion gradients.