Spatial and temporal changes of intracellular calcium ion concentration ([Ca2+]i) during stimulated contractions were observed by confocal microscopy in rat ventricular and guinea pig atrial myocytes. Fluorescence intensity profiles in fluo 3-acetoxymethyl ester (fluo 3-AM)-loaded cells were collected from the entire cell, selected regions of the cell, or along a single scanned line across the cell. In rat ventricular myocytes, the increase of [Ca2+]i after a single stimulus from field electrodes occurred synchronously across the cell whether fluo 3 fluorescence was monitored in a narrow region aligned with the long axis of the cell or in line-scan images of a single z-line across the cell. However, during the onset of Ca2+ channel blockade by nifedipine (5 microM), electrical stimulation produced spatially nonuniform, focal increases of [Ca2+]i. In guinea pig atrial myocytes, stimulated increases of [Ca2+]i first appeared in focal regions at the cell periphery before spreading to the cell interior. Line-scan images showed the peripheral rise of [Ca2+]i led that at the center of the cell by 34 +/- 4 ms (mean +/- SE, n = 3). These data demonstrate that the t-tubular network ensures synchronous increases of [Ca2+]i throughout the cell during an action potential. In the absence of t tubules or when the number of sarcolemmal Ca2+ channels opened by membrane depolarization is greatly reduced, stimulated increases of [Ca2+]i can be observed to arise in focal regions of the cell.