Atrial (ANP) and B-type natriuretic peptides (BNP) modulate blood pressure and volume through the stimulation of cyclic GMP production by their guanylyl cyclase-A (GC-A) receptor. A novel isoform of GC-A has been identified that is the result of differential splicing of exon 4. The deletion of a 51-bp sequence is predicted to delete 17 amino acids (Lys314-Gln330) in the membrane-distal part of the extracellular domain. Reverse transcription-PCR analyses demonstrated low messenger RNA expression levels of spliced GC-A in all tissues. Homology modeling suggested that the alterations in the protein structure could interfere with ANP binding or signaling. Indeed, functional studies in transfected HEK 293 cells demonstrated that binding of ANP and ANP-induced cyclic GMP formation by GC-ADelta(Lys314-Gln330) were totally abolished. Furthermore, cotransfection studies showed that this GC-A variant forms heterodimers with the wild type receptor and inhibits ligand-inducible cGMP generation. Finally, treatment of mice with angiotensin II (300 ng/kg/min during 7 days) resulted in enhanced pulmonary mRNA expression of spliced GC-A, which was concomitant to diminished GC-A/cGMP responses to ANP. We conclude that alternative splicing can regulate endogenous ANP/GC-A signaling. Angiotensin II-induced alternative splicing of GC-A may represent a novel mechanism for reducing the sensitivity to ANP.