1. The origins of local [Ca2+]i transients (Ca2+ sparks) were studied using dual-channel confocal laser scanning microscopy. Line scan images showing [Ca2+]i (as fluo-3 fluorescence) and the transverse tubule membranes (as Di-8 fluorescence) were obtained simultaneously in single rat cardiac ventricular cells. 2. Line scan images of Di-8 fluorescence showed peaks regularly spaced at intervals of 1.83 +/- 0.30 microns (mean +/- S.D.). These peaks corresponded to the transverse tubules (T-tubules) in cross-section. 3. Line scan images of fluo-3 fluorescence showed local [Ca2+]i transients (LCTs or Ca2+ sparks) evoked by electrical stimulation. 4. Eighty-five per cent (85%) of all Ca2+ sparks evoked by electrical stimulation (n = 138, in 5 cells) occurred within 0.5 micron of a T-tubule. Thirty per cent (30%) occurred within 1 pixel (0.20 micron) of a T-tubule. 5. In some cells studied (3 out of 5), certain T-tubules had a higher probability of being sites of origin of Ca2+ sparks than others. 6. These results support local control theories of excitation-contraction coupling in which Ca2+ release from the sarcoplasmic reticulum (SR) is triggered by a high local [Ca2+]i established between the L-type Ca2+ channels in the T-tubules and associated ryanodine receptor(s) in the junctional SR.