Ageing is associated with a decline in cardiac function (1). The L-type calcium current (ICa), which triggers intracellular Ca release and thus contraction, has been reported to decrease or to remain unchanged with age (2,3). However, the effect of age on the localisation of ICa to the t-tubules, invaginations of the surface membrane of ventricular myocytes where the majority of Ca influx and release normally occurs, is unknown. We have, therefore, investigated ICa density in the t-tubular and surface membranes of ventricular myocytes isolated from 3 month (3 mo) and 24 month (24 mo) old mice. Animal procedures were approved by local ethics committee and conducted in accordance with UK legislation. Ventricular myocytes were isolated by enzymatic digestion of hearts excised from male C57BL/6 mice. ICa was recorded using the whole-cell patch-clamp technique in intact cells and following acute detubulation (DT, using formamide-induced osmotic shock (4)), at 22-25°C. ICa was elicited by a 500 ms step depolarisation from -40 to 0 mV following a 200 ms pre-pulse from -80 to -40 mV, at 0.2Hz and was measured as the difference between peak current and that at the end of the pulse. ICa recorded from DT myocytes represents ICa at the surface membrane and the difference in ICa between intact and DT myocytes gives ICa at the t-tubule membrane. Data are expressed as mean±SEM (n cells). Student’s t-test or ANOVA were used for statistical analysis, with the Bonferroni post hoc test; the limit of statistical confidence was p<0.05. ICa amplitude was not significantly different in myocytes isolated from 3 mo and 24 mo old mice (3 mo, 1.4±0.1 (n=27); 24 mo, 1.2±0.8 (n=25), nA at 0mV) but cell capacitance increased with age (3 mo, 217±11; 24 mo, 269±12, pF, p<0.001) so that ICa density was significantly smaller in the 24 mo myocytes (3 mo, -6.4±0.4; 24 mo, -4.6±0.3, pA/pF at 0mV, p<0.001) without a change in the rate of ICa inactivation. DT caused a greater decrease in ICa density in myocytes from 3 mo mice than in cells from 24 mo mice (p<0.05), so that ICa density in DT myocytes (at the surface membrane) was not significantly different in myocytes isolated from 3 mo and 24 mo hearts, but ICa density at the t-tubule membrane decreased from -14.5±0.5 (3 mo) to -7.1±0.3 pA/pF (24 mo, p<0.001). These data show that the distribution of ICa is different in myocytes from 3 mo and 24 mo old mice, with ICa density decreasing by ~50% at the t-tubule membrane with no change at the surface membrane, so that t-tubule:surface ICa density decreases from ~5 to ~2.8, in aged myocytes, which may contribute to altered excitation-contraction coupling with age.