β-adrenergic stimulation in cardiac myocytes is mainly due β1- and β2-receptors (ARs). The sub-cellular distribution of specific β-ARs is unclear. Immunocytochemistry data have shown that key proteins of the β-adrenergic pathway concentrated in the t-tubules (TT) of cardiac ventricular myocytes (versus surface sarcolemma (SS) [1]. However, a recent report suggested that only β2-ARs were located in the TT (and not at the SS), whereas β1-ARs are evenly distributed between TT and SS [2]. Nevertheless, this study only recorded the increase in cyclic AMP (cAMP) during β-adrenergic stimulation and not the functional effect of such stimulation. Therefore, the functional distribution of β-ARs in ventricular cardiac myocytes (TT versus SS) is still unclear. This study addresses this point. Rat ventricular cells were enzymatically isolated according to the UK law. Detubulation was achieved using osmotic shock as previously described [3]. Intracellular calcium concentration was recorded using fluorescent dye (fura-2 AM) and cell contraction was induced by field stimulation. Selective β1-adrenergic stimulation was achieved by perfusion of isoprenaline (0.1 μM) and ICI 118,551 (0.1 μM). Selective β2-adrenergic stimulation was achieved by perfusion of salbutamol (10 μM) and atenolol (1 μM). In control cells, β1-adrenergic and β2-adrenergic stimulation caused a significant increase in peak calcium transient (peak CaTr; 236.8±42.9%, n=29 and 24.6±4.2%, n=41, respectively), evaluating full β-adrenergic stimulation (i.e. SS + TT). In detubulated cells, β-adrenergic stimulation had a greater effect on peak CaTr than in control cells (288.1±80.6% increase for β1, n=17 and 83.5±19.0% for β2 n=20; evaluating β-adrenergic stimulation only from SS). From these values, we calculated that the % of increase of peak CaTr from the TT was ~128.3% during β1-adrenergic, and ~0.88% during β2-adrenergic. These data clearly indicates that both β1- and β2-pathways are functional in SS and TT in ventricular cardiac cells. In addition, we found that β2-adrenegic stimulation is only functional (i.e. has an affect on CaTr) at the TT, suggesting that β2-adrenegic stimulation has a physiological effect via the SS. These results are in contradiction with the latest report about the localisation of β-adrenergic receptors [2]. However, our study focuses on the functional β-ARs response (i.e. response in calcium concentration and CS) instead of the response to the increase of cyclic AMP, which may account for the different conclusion.