In the myocyte, the Ca transient determines the force and speed of contraction and therefore tight control of Ca movement is paramount to normal physiology. The Ca transient is controlled by autoregulation [1]; the balance between Ca influx and Ca efflux which occur mainly via L-type Ca channels (LTCC) and Na/Ca exchanger (NCX) respectively. LTCCs [2] and NCX [3] are predominantly located in t-tubules, and thus autoregulation is hypothesised to occur predominately within these structures [4]. Caveolin-3 (Cav3) is a scaffolding protein associated with t-tubule formation in myocytes [5]. This study therefore aims to investigate the consequence of Cav3 knock out (KO) on autoregulation in mouse ventricular myocytes. Myocytes were isolated from 12 week old male Cav3 KO and their littermate controls by enzymatic digestion. Cells were field stimulated at 1Hz and the Ca transients were monitored using Fura-2 AM, and reported as the ratio between the fluorescence excited at 340nm and 380 nm (F340/380). Autoregulation was investigated during the application of 200 µM caffeine, increasing ryanodine receptor (RyR) opening probabilities, and following the application of 10 mM caffeine, to empty the sarcoplasmic reticulum (SR). Data are presented as mean±SEM and compared by mixed model ANOVA and Sidak post hoc test. Cav3 KO myocytes showed no significant difference in steady-state Ca transient amplitude compared to littermate controls [F340/380 0.17±0.03 (KO, n=19) vs 0.17±0.01 (WT, n=12)] and the application of 200 µM caffeine produced similar increase in transient amplitude in both cell-types [F340/380 0.26±0.05 (KO) vs 0.27±0.03 (WT)]. During the continued presence of 200 µM caffeine, the transient amplitude recovered to steady-state in both cell-types, however the time taken to achieve this was significantly increased in the Cav3 KO cells [48±2.82 (KO) vs 30±2.12 s (WT), p<0.0001). The increase in fluorescence during the application of 10 mM caffeine, a measure of SR Ca content, was not significantly different in Cav3 KO cells compared to WT controls [F340/380 0.40±0.03 (KO, n=10) vs 0.41±0.04 (WT, n=10)] and no significant difference was observed in the rate of decay of the caffeine-induced transient [0.35±0.03 (KO) vs 0.38±0.02 s-1 (WT)]. This would suggest that NCX activity is similar in both cell-types. Following washout of 10 mM caffeine, stimulation was resumed, and Ca transient amplitudes, which were initially small, recovered to steady state. As with the application of 200 µM caffeine, the time taken to reach steady state was significantly increased in Cav3 KO cells [15 ±1.44 (KO) vs 10±1.44 s (WT), p<0.01]. These data suggest that autoregulation is slower in Cav3 KO mice, which is unlikely to be due to changes in NCX.