The transverse (t-) tubules of mammalian cardiac ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell. The function of many of the key proteins involved in cardiac excitation-contraction coupling occurs predominantly at the t-tubules: acute detubulation results in an ~80% decrease of L-type Ca current (ICa), an ~65% decrease of Na-Ca exchange current (INCX), and complete loss of Ca extrusion via the sarcolemmal Ca ATPase (see Orchard et al., 2009 for review; Chase & Orchard, 2011). Thus the majority of trans-sarcolemmal Ca influx and efflux occurs across the t-tubule membrane, which ensures that both activation and relaxation occur rapidly throughout the cell. Cell signalling is also different at the t-tubule and surface membranes: the β-adrenergic agonist isoprenaline causes greater stimulation of ICa at the t-tubules than at the surface membrane, suggesting better coupling of the β -adrenergic pathway to ICa at the t-tubules (Brette et al., 2004), and recent work suggests that localisation of ICa and INCX to the t-tubules is due, in part, to localised stimulation of these currents at the t-tubules by basal activity of protein kinase A (PKA; Chase et al., 2010; Chase & Orchard, 2011). Since β2 adrenoceptors occur predominantly in the t-tubules in normal ventricular myocytes (Nikolaev et al., 2010), it seems possible that tonic activity of the β2 adrenergic pathway underlies this localisation. To investigate further the mechanisms underlying this localisation, we have used a membrane-permeable peptide representing the scaffolding domain of Caveolin-3 (Cav-3; MacDougall et al., 2012). This peptide (C3SD) reduced basal ICa and inhibited the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of H-89 and C3SD on basal ICa were not summative, suggesting that they share a common pathway involving PKA. Thus Cav-3 appears to play an important role in facilitating stimulation of ICa by basal PKA activity and in response to β2-adrenoceptor stimulation, consistent with the hypothesis that basal activity of the β2-adrenergic pathway underlies stimulation of ICa, to which it is coupled via Cav-3. T-tubules may also play an important role in pathological conditions. Changes in t-tubule morphology have been reported in both human heart failure (HF) and in animal models of HF, with t-tubule structure becoming disordered, with consequent changes in the synchrony of Ca release. Changes in cell signalling also occur: H-89 has a greater inhibitory effect on basal ICa in myocytes isolated 16 weeks after coronary artery ligation than in those from sham operated controls, suggesting greater stimulation by PKA, as shown previously. However, C3SD does not decrease ICa significantly in the ligation model (Bryant et al., 2013), suggesting that the coupling of PKA activity to ICa by Cav-3 described above is already disrupted. Although the role of the t-tubules in these changes is still being elucidated, it is interesting that HF is associated with redistribution of β2 adrenoceptors from the t-tubules to the surface membrane, and a change from localised to diffuse cAMP signalling in response to β2 adrenoceptor stimulation (Nikolaev et al., 2010), making it tempting to speculate that loss of Cav-3 coupling plays a role in these changes. Thus the t-tubules appear to form a specialised domain for trans-sarcolemmal Ca flux and its regulation, and thereby play a key role in physiological and pathophysiological cell function.