Abstract—The [Ca2+]itransient of ventricular myocytes during normal excitation-contraction coupling is the summation of primary Ca2+release events, which originate at the junction of the sarcoplasmic reticulum (SR) and the T-tubular system. Studies in small mammals have shown a high density of release sites, but little is known of larger mammals. We have studied the spatial distribution of SR Ca2+release in pig ventricular myocytes using a confocal microscopy. In 69 of 107 cells, large inhomogeneities of Ca2+release were observed along the longitudinal scan line. Areas where the increase of [Ca2+]iwas delayed (time to 50% of peak F/F0[where F indicates fluorescence intensity, and F0indicates F at rest] was 26±1 ms in delayed areas versus 11±2 ms in early areas) and smaller (peak F/F0was 2.27±0.10 for delayed areas versus 2.69±0.13 for early areas; n=13 cells,P<0.05) could be up to 26 &mgr;m wide. The sum of all delayed areas could make up to 55% of the line scan. The spatial pattern was constant during steady-state stimulation and was not altered by enhancing Ca2+channel opening or SR Ca2+content (Bay K8644, isoproterenol). Imaging of sarcolemmal membranes revealed several areas devoid of T tubules, but SR Ca2+release channels were homogeneously distributed. In contrast, compared with pig myocytes, mouse myocytes had a very dense T-tubular network, no large inhomogeneities of release, and a faster rate of rise of [Ca2+]i. In conclusion, in pig ventricular myocytes, areas of delayed release are related to regional absence of T tubules but not ryanodine receptors. This lower number of functional couplons contributes to a slower overall rate of rise of [Ca2+]i.