Immunocytochemistry data suggest that neuronal Na channels are present at the transverse (t-) tubules of cardiac ventricular myocytes (Maier et al. 2002); however, no data about the distribution of function (i.e. current) are available. In the present study, we recorded Na current (I_Na) in control and detubulated myocytes (Brette et al. 2002), which enabled us to determine and quantify the functional localization of specific isoforms of I_Na in rat ventricular myocytes. Wistar rats were killed humanely. Ventricular myocytes were enzymatically isolated, and detubulated using formamide as described by Kawai et al. (1999). The whole-cell configuration of the patch clamp technique was used to record I_Na. To ensure good voltage control, extracellular [Na] was decreased to 20 mM, low resistance pipettes were used (1.5 ± 0.1 MΩ, mean ± S.E.M., n=32) and series resistance (2.8 ± 1.1 MΩ, n=32) was compensated by 85-90%. I_Na was elicited by a test pulse to −40 mV from a holding potential of −120 mV. Neuronal I_Na was assessed as the current sensitive to a low (100 nM) concentration of TTX (Maier et al. 2002). Experiments were performed at room temperature (22-24°C). Cell capacitance was significantly smaller in detubulated cells than in control cells (in pF: 137 ± 7 vs 199 ± 9, n=15 and 17, respectively, P<0.05, two tailed unpaired t test); however, I_Na density (in pA/pF: 73 ± 5 (control) vs 81 ± 8 (detubulated), NS) and time to peak (in ms: 1.39 ± 0.08 (control) vs 1.42 ± 0.07 (detubulated) NS) were unchanged. 100 nM TTX blocked 24 ± 2% of I_Na in control myocytes and 8 ± 2% in detubulated myocytes, suggesting that more neuronal I_Na (I_NaN) is present in control, than in detubulated myocytes (P<0.05, unpaired t-test). From these data, we calculated the relative distribution of currents as previously described (Despa et al. 2003). The cardiac I_Na (I_NaC, residual I_Na during application of 100 nM TTX) appears to be almost exclusively present at the surface membrane (95%) where it is 10 times more concentrated than in the t-tubules. In contrast, I_NaN is located predominantly in the t-tubules (75%) where it is 6.4 times more concentrated than at the surface membrane. We conclude that although I_Na is uniformly distributed between the t-tubules and surface membrane of rat ventricular myocytes, this masks a marked differential localization of I_Na isoforms, which suggests specific physiological roles for I_NaC (e.g. excitability & conduction) and I_NaN (e.g. t-tubule excitability).