Atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth and stimulates angiogenesis by a transmembrane guanylyl cyclase receptor (GC-A) and presumably by the second messenger cyclic GMP. Recently we observed that ANP can stimulate TRPC-mediated Ca²⁺ entry in cardiomyocytes in GC-A-dependent but cGMP-independent manner (see Abstract by M. Klaiber et al.). Here we examined the possibility of a direct interaction between GC-A and TRPC3. TRPC3 was coexpressed in HEK293 cells together with FLAG-tagged GC-A and the membrane fraction was subjected to immunoprecipitation with anti-FLAG antibody. Western blot analyses demonstrated the co-immunoprecipitation of TRPC3 with GC-A in presence and absence of ANP. This ANP-independent interaction was also observed with a truncated ANP receptor lacking the cGMP-forming guanylyl cyclase domain. To validate the interaction of GC-A and endogenous TRPC3 in cardiac myocytes, we generated a transgenic mouse model with cardiomyocyte specific overexpression of HA-tagged GC-A. Immunoprecipitation of GC-A by anti-HA antibody demonstrated its association with native TRPC3. Finally, to study how closely GC-A and TRPC3 interact with each other, we used a FRET-based approach. Coexpression of GC-A-CFP and TRPC3-YFP resulted in a substantial FRET signal, corroborating close proximity between their C termini. ANP (1–100 nM) induced rapid and concentration-dependent decreases in FRET within ~10 sec, indicative of agonist-induced conformational changes. FRET signals were insensitive to the membrane-permeable cGMP analog 8-Br-cGMP (100 mM, 10 min). Our results reveal a novel cGMP-independent signaling pathway of GC-A, which involves a stable protein complex of GC-A and the TRPC3 channel. Supported by SFB 487.