Guanylyl cyclase-B (GC-B) is a single transmembrane receptor that binds C-type natriuretic peptide (CNP). The ligand/receptor appears critical in the regulation of cell proliferation and differentiation where it acts as an adversary of mitogenic signaling pathways. We have isolated three guanylyl cyclase-B isoforms generated from a single gene by alternative splicing and termed them GC-B1, GC-B2, and GC-B3. GC-B1 is full-length and responds maximally to CNP, GC-B2 contains a 25-amino acid deletion in the protein kinase homology domain, and GC-B3 only retains a part of the extracellular ligand-binding domain. GC-B2 binds CNP, but the ligand fails to activate the cyclase, while GC-B3 fails to bind ligand. When GC-B2 or GC-B3 is expressed coincident with GC-B1, they act as dominant negative isoforms by virtue of blocking formation of active GC-B1 homodimers. Relative expression levels of GC-B1, GC-B2, and GC-B3 vary across tissues and as a function of in vitro culture; the relative amount of GC-B2 to GC-B1 is repressed in cultured smooth muscle cells relative to endogenous ratios in the medial layer cells of the aorta. Thus, GC-B isoform levels can be independently regulated. Given that the splice variants serve as dominant negative forms, these will serve as regulators of the full-length GC-B.