Intracellular Ca in cardiac myocytes is regulated by the balance between Ca influx and Ca efflux (1). In ventricular myocytes, the main Ca influx pathway, L-type Ca current (ICa), is predominantly located in the t-tubules (TT) (2), and data from rat indicate that the main Ca efflux pathway, Na-Ca exchange current (INCX), is also greater at the TT than at the surface sarcolemma (SS) (3). However the distribution of INCX in mouse myocytes, and the role of t-tubular ICa and INCX in Ca autoregulation, are unknown. Animal procedures were performed in accordance with UK legislation. Mouse ventricular myocytes were isolated by enzymatic digestion, and detubulated (DT) by osmotic shock (3). Membrane current recordings were made from intact and DT myocytes at room temperature using the whole-cell voltage-clamp technique. Intracellular Ca concentration was monitored simultaneously using fluo-4. Sarcoplasmic reticulum (SR) Ca release was triggered using caffeine (10 mM) and the associated INCX recorded at -80 mV. Following washout of caffeine, pulses to 0 mV were applied at 1 Hz to activate ICa and trigger a Ca transient (CaT); Ca influx during each pulse was measured as the integral of ICa at 0 mV and Ca efflux as the integral of INCX upon repolarisation to -80 mV (1). Data are presented as mean±SEM of n cells and statistical significance determined by Student’s t-test (p<0.05 was the limit of confidence). There was no significant difference in caffeine-induced Ca transient amplitude between intact and DT myocytes [F/F0: 3.16±0.26 (intact; n=13) vs 3.06±0.54 (DT; n=10)] or in the associated peak INCX density [-1.03±0.09 vs -1.18±0.13 pA/pF respectively], suggesting equal INCX density in TT and SS. Recovery of CaT amplitude following washout of caffeine reflects restoration of SR Ca content to steady state (autoregulation). Recovery was slower [half-time 8.7±1.0 (intact) vs 12.6±0.4 beats (DT), P<0.001] and steady-state CaT amplitude was reduced [F/F0 2.80±0.28 (intact) vs 1.57±0.16 (DT), P<0.01] in DT compared to intact cells. Ca influx and efflux were both smaller in DT than in intact cells, but recovery of CaT amplitude to steady-state was associated with a reduction in Ca influx of approximately 15% in both cell types, from -108.8±10.5 to -91.3±8.0 pA.s/pF (intact) and from -65.3±4.4 to -56.6±3.5 pA.s/pF (DT), However, Ca efflux increased 4-fold during recovery of the CaT in intact cells from -15.1±1.5 to -59.2±6.7 pA.s/pF, but only 2-fold in DT cells from -14.9±2.9 to -26.0±3.5 pA.s/pF. These data suggest that INCX density is similar in the SS and TT in mouse myocytes, and is the main mechanism for autoregulation (4), and that loss of Ca fluxes across the TT membrane slows recovery following DT.