In cardiac myocytes, the scaffolding protein caveolin-3 (Cav-3) plays an important role in determining membrane structure and in localizing the function of key proteins, including the L-type Ca channel, and thereby L-type calcium current (ICa), to the t-tubules. Acute inhibition of Cav-3 binding to endogenous proteins decreases ICa density preferentially at the t-tubules resulting in more uniform distribution of ICa between the t-tubule and surface membranes. Heart failure is associated with decreased Cav-3 levels (1) and redistribution of ICa away from t-tubules (2). We have, therefore, compared the effect of caveolin-3 knockout (Cav-3 KO) and transverse aortic constriction (TAC) on ICa distribution in mice. Animal procedures were approved by local ethics committee and conducted in accordance with UK legislation. Ventricular myocytes were isolated from: (i) 20 week-old male C57BL/6 mice that had been subjected to TAC at 12 weeks, which were compared with sham-operated and age matched controls; mice undergoing surgery were given analgesia (buprenorphine 0.1 mg/kg s.c) and anaesthetised with ketamine (75 mg/kg i.p.) and medetomidine (0.5 mg/kg i.p.). (ii) 12 week-old Cav-3 KO mice (3) which were compared with wild-type controls. The whole-cell patch-clamp technique was used to record ICa from intact ventricular myocytes, and following acute detubulation (DT) using formamide-induced osmotic shock (4), at 22-25°C. ICa recorded from DT myocytes represents ICa at the surface membrane. Data are expressed as mean±SEM (n cells). Student’s t-test or ANOVA were used for statistical analysis, post hoc test used Bonferroni correction; the limit of statistical confidence was p<0.05. Cellular hypertrophy was observed in TAC and Cav-3 KO mice as an increase in cell volume (pl: sham 57.6 ± 3.4 (24) vs TAC 69.5 ± 3.6 (16), p<0.03; WT 43.4 ± 2.7 (19) vs KO 60.8 ± 3.8 (17), p<0.001) and cell capacitance (pF: sham 213 ± 9 (24) vs TAC 308 ± 15 (16), p<0.001; WT 169 ± 6 (19) vs KO 228 ± 11 (17), p<0.001). ICa density was decreased in intact TAC and KO myocytes (pA/pF: sham -7.1 ± 0.3 (24) vs TAC -6.3 ± 0.2 (16), p=0.05; WT -7.6 ± 0.4 (19) vs KO -6.1 ± 0.5 (17), p<0.05). In DT myocytes, ICa density was unchanged by either TAC or KO (pA/pF: sham -3.6 ± 0.2 (26) vs TAC -3.6 ± 0.2 (19), ns; WT -3.8 ± 0.3 (22) vs KO -3.9 ± 0.3 (20), ns). It follows that ICa density at the t-tubule membrane decreased in both TAC and KO myocytes. In conclusion, these data show that cardiac hypertrophy/failure and Cav-3 KO are both associated with cellular hypertrophy and a decrease in ICa density at the t-tubule membrane with no change at the cell surface. Thus the decrease of Cav-3 expression that occurs in heart failure may underlie these changes.