Cardiac L-type Ca current (ICa) is the primary trigger for Ca release from sarcoplasmic reticulum (SR) and the major influx pathway maintaining cell Ca load and contractility. In normal ventricular myocytes, ICa is localised predominantly to t-tubules (TT) but its relocation to the surface sarcolemma (SS) may contribute to systolic dysfunction in heart failure. The aim of this study was to investigate the effect of this relocation on Ca loading. Animal procedures were performed in accordance with UK legislation and approved by local ethics committee. Coronary artery ligation (CAL) and sham operations (Sham) were performed on adult male Wistar rats under surgical anaesthesia (ketamine 75 mg/kg, medetomidine 0.5 mg/kg ip) with appropriate analgesia (buprenorphine 0.05 mg/kg sc). Hearts were excised under pentobarbitone anaesthesia (140 mg/kg ip) 18 weeks after surgery and left ventricular myocytes isolated by enzymatic digestion. Cells were voltage-clamped to -80 mV using whole-cell patch-clamp. Following a ramp to -40 mV ICa was recorded at 0 mV. The pipette contained 100 mM fluo-4 for simultaneous measurement of Cai. Cells were stimulated at 1 Hz for 20 s followed by a 10 s gap before 10 mM caffeine was applied for 10 s to release SR Ca. 1 Hz stimulation was resumed 10 s after removal of caffeine. Recordings were made at room temperature from intact cells (Sham n=12, CAL n=8) and cells detubulated by osmotic shock (DT, Sham n=12, CAL n=7) to calculate SS and TT ICa densities. Data are presented as mean±SEM and analysed using 2-way ANOVA with Bonferroni post-test; the limit of statistical confidence was P<0.05. In all cells, Cai transient amplitude on the 1st stimulus after caffeine was smaller than steady-state and increased with repeated stimulation. However, there was no significant difference in the rate of recovery in Sham and CAL cells, and DT had no significant effect on recovery rate in either group. SS ICa density during the 1st beat after caffeine was not significantly different in Sham and CAL cells (Sham -2.98±0.26, CAL -3.31±0.31 pA/pF), and decreased during recovery from caffeine (P<0.01) to steady-state values that were not significantly different (Sham -2.42±0.21 pA/pF; CAL -2.50±0.26 pA/pF), consistent with accumulation of inactivation of SS ICa. In contrast, TT ICa density was markedly smaller in CAL cells during the 1st beat after caffeine (Sham -5.92±0.32, CAL -0.91±0.37 pA/pF, P<0.001), and increased by a similar amount in both groups of cells during recovery (Sham -6.85±0.27 pA/pF P<0.001, CAL -1.84±0.33 pA/pF P<0.001), indicating facilitation of TT ICa. These data provide evidence for differential acute regulation of ICa at SS and TT, and suggest that SS plays a key role in Ca loading.