TRPC proteins form Ca²⁺-permeable cation channels activated following stimulation of G protein-coupled receptors linked to phospholipase C (PLC). Although the PLC/inositol-phosphate signaling pathway is known to exist in heart, the expression and subcellular distribution of TRPC channel proteins in ventricular myocardium has not been evaluated. Of the six members of the TRPC channel family examined in the present study, only TRPC3 was found by Western blot analysis of membrane proteins from either rodent or canine ventricle. Likewise, only TRPC3 was observed in immunofluorescence analysis of thin sections from rat ventricle or in neonatal rat ventricular myocytes in culture. In longitudinal sections of rat ventricle, TRPC3 was predominantly localized to the intercalated disk region. However, transverse sections through heart muscle or single isolated adult myocytes revealed TRPC3 labeling in a vast network of intracellular membranes where it co-localized with the Na⁺,K⁺-ATPase (NKA) pump and the Na⁺,Ca²⁺-exchanger (NCX), but not with the ryanodine receptor or the sarcoplasmic-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump. Immunoprecipitation assays from rat or canine ventricle showed that TRPC3 associates with NKA and NCX, but not with the plasmalemmal Ca²⁺-ATPase (PMCA) pump or SERCA. Immunoprecipitations from Sf9 insect cells expressing TRPC3, NKA, and NCX in various combinations revealed that NKA and NCX interact, and that TRPC3 and NCX interact, but that TRPC3 does not directly associate with the NKA. Together, these results suggest that TRPC3 is localized in the ventricular myocyte to the axial component of the transverse-axial tubular system (TATS) where it exists in a signaling complex that includes the NCX and the NKA.