Ca²⁺ influx into cardiac ventricular myocytes, via the L-type Ca²⁺ current, appears to occur mainly across the t-tubule membrane. Ca²⁺ efflux, via Na+/Ca2+ exchange (NCX), also appears to occur predominantly, but not exclusively, across the t-tubule membrane (1). However the location of the sarcolemmal Ca²⁺ ATPase is unknown. We have therefore investigated the distribution of Ca²⁺ efflux via the Ca²⁺ ATPase between the t-tubule and surface membranes. Ventricular myocytes were isolated from the hearts of male Wistar rats, and detubulated as described previously (2). Intracellular Ca²⁺ was recorded using fluo-3 in conjunction with confocal microscopy during electrical stimulation at 0.5 Hz. Following a train of stimuli, 20 mM caffeine was used to release Ca²⁺ from the sarcoplasmic reticulum (SR); this was repeated in the presence of 10 mM NiCl, to inhibit NCX, or following 8-10 minutes incubation with 20 μM carboxyeosin, to inhibit the sarcolemmal Ca²⁺ ATPase, in control and detubulated myocytes. The proportion of Ca²⁺ removed from the cytoplasm by different pathways was calculated from the rate constants of decline of the electrically stimulated Ca²⁺ transient, and those obtained in the presence of caffeine, as described previously (3). The rate constant of decline of the systolic Ca²⁺ transient was 2.985±0.254^s-1 in control myocytes, and 2.8±0.218^s-1 in detubulated myocytes (n=22/26; p=0.5810; unpaired t-test). The rate of decline of the caffeine-induced Ca²⁺ transient was slower: 0.520±0.115^s-1 in control myocytes and 0.188±0.031^s-1 in detubulated myocytes (n=9/11; p=0.0109); this was decreased further by NiCl, from 0.520±0.115 to 0.179±0.022^s-1 in control myocytes (n=9/12; p=0.0057), and from 0.188±0.031 to 0.083±0.012^s-1 in detubulated myocytes (n=11/12; p=0.0212). The rate of decline of the caffeine-induced Ca²⁺ transient was also slowed by carboxyeosin in control myocytes, from 0.520±0.115 to 0.294±0.047^s-1 (n=9/9; p=0.0484), but not in detubulated myocytes (0.188±0.031 vs. 0.203±0.36^s-1; n=11/7; ns). In control myocytes, therefore, the SR appears to be responsible for ~83% of Ca²⁺ removal, NCX for ~11%, and the sarcolemmal Ca²⁺ ATPase for ~7%, while in detubulated myocytes, the SR is responsible for ~93%, NCX for ~4% and the Ca²⁺ ATPase for 0%. Thus Ca2+ efflux via the sarcolemmal Ca²⁺ ATPase appears to occur only across the t-tubule membrane, so that the slower Ca²⁺ extrusion in detubulated cells is likely to be due to partial loss of NCX and complete loss of Ca²⁺ ATPase.