Transverse (t-) tubules are invaginations of the surface membrane of mammalian cardiac ventricular myocytes. The function of many of the key proteins involved in excitation-contraction coupling occurs predominantly at the t-tubules; it has been suggested that this is due, in part, to localised stimulation of ion flux pathways at the t-tubules by protein kinase A (PKA)-induced phosphorylation. Caveolin-3 (Cav-3) has previously been implicated in the localisation of PKA activity. We have, therefore, investigated the role of Cav-3 on the L-type Ca current (ICa), which flows predominantly across the t-tubule membrane, in rat ventricular myocytes. Myocytes were isolated from male Wistar rats (250-300 g). Animals were killed either by cervical dislocation or under pentobarbitone (140 mg/kg i.p.) general anaesthesia in accordance with UK legislation. ICa was recorded using the whole-cell patch clamp technique with 5 mM BAPTA in the pipette solution to inhibit Ca-dependent inactivation of ICa. 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 (-50 to +80 mV) to activate ICa (0.2 Hz at room temperature). Standard immunohistochemical techniques were used in conjunction with confocal microscopy to determine the localisation of Cav-3, L-type Ca channels (LTCC) and phosphorylated LTCC (pLTCC). Incubation with TAT-tagged C3SD peptide (C3SD) was used to disrupt normal protein binding to Cav-3 (MacDougall et al., 2012). Scrambled peptide (Scram) and non incubated cells (Con) were used as controls. β2-adrenoceptor stimulation was achieved by zinterol (Zint, 1-10 µM) applied to cells pre-exposed to atenolol (10 µM). Data are expressed as mean±SEM (n). 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. The amplitude of ICa at 0 mV was not altered by Scram but was reduced by C3SD (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). C3SD had no effect on the distribution of staining of Cav-3 or LTCC but significantly altered the distribution of pLTCC staining. The PKA inhibitor H-89 decreased the amplitude of ICa at 0 mV in the absence and presence of C3SD (Con -7.6±0.5 to -3.6±0.5 (5) pA/pF, p<0.001; C3SD -5.4±0.4 to -3.7±0.3 (5) pA/pF, p<0.001). Peak ICa was not different between the 2 groups in the presence of H-89. H-89 also decreased staining of pLTCC. The concentration-dependent increase of ICa caused by Zint was inhibited in cells incubated in C3SD. These data are compatible with the hypothesis that Cav-3 plays an important role in mediating the stimulation of ICa by PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation.