In cardiac ventricular myocytes many of the key proteins involved in excitation-contraction coupling, including L-type calcium channels (LTCC), are located predominantly at the transverse (t-) tubules. Protein kinase A (PKA)-mediated stimulation of L-type Ca current (ICa) also occurs predominantly at the t-tubules. Caveolin-3 (Cav-3) has been implicated in the localization of PKA activity to macromolecular complexes. We have, therefore, investigated whether Cav-3 plays a role in the stimulation of ICa by PKA in the t-tubules of rat ventricular myocytes. Ventricular myocytes were isolated from the hearts of male Wistar rats (250-300 g) killed either by cervical dislocation or under pentobarbitone (140 mg/kg i.p.) anaesthesia in accordance with UK legislation. ICa was recorded using the whole-cell patch-clamp technique. From a holding potential of -80 mV, a 100 ms step depolarisation to -40 mV was used to inactivate INa, followed by a 500 ms step depolarisation (to voltages between -50 and +80 mV) to activate ICa (0.2 Hz; room temperature). Standard immunohistochemical techniques were used in conjunction with confocal microscopy to determine the localization of Cav-3, total LTCC (tLTCC) and phosphorylated LTCC (pLTCC). Incubation with TAT-tagged C3SD peptide (C3SD) was used to disrupt normal Cav-3 protein binding (MacDougall et al., 2012). Scrambled peptide (Scram) and non-incubated cells (Con) were used as controls. β2-adrenoceptor stimulation was achieved using zinterol (1-10 µM) applied to cells pre-exposed to atenolol (10 µM). Detubulation (DT) was achieved by exposing cells to 1.5M formamide (Kawai et al., 1999). Data are expressed as mean±SEM (n cells). Statistical analysis was performed by analysis of variance (1 or 2 way) with the appropriate Bonferroni post hoc test; significance was taken at p<0.05. In control cells, ICa density was significantly reduced by C3SD and unaffected by Scram peptide (Con -7.6±0.3 (11), Scram -8.0±0.4 (9), C3SD -5.4±0.2 (15) pA/pF, p<0.0001 C3SD vs Scram). However, in detubulated cells C3SD did not decrease ICa (DT -4.7±0.2 (5), DT+C3SD -4.2±0.4 (6) pA/pF, ns). C3SD had no effect on the distribution of staining of Cav-3 or tLTCC but significantly altered the distribution of pLTCC staining. The PKA inhibitor H-89 decreased ICa by 53±3% in Con, but by only 28±10% in the presence of C3SD (p<0.001), to a level that was not significantly different from in the presence of H-89 alone (Con -3.6±0.5 (5), C3SD -3.7±0.3 (5) pA/pF, ns). The increase of ICa induced by zinterol was inhibited in cells incubated with C3SD. These data are consistent with the notion that Cav-3 plays an important role in mediating the localised stimulation of t-tubular ICa by basal PKA activity and in response to β2-adrenoceptor stimulation.