Atrial natriuretic peptide (ANP) binds directly to a plasma membrane form of guanylate cyclase (GC-A), stimulating the production of the second messenger cyclic GMP. We show that a second guanylate cyclase/receptor (GC-B) exists, with distinctly different specificities for various natriuretic peptides. A cDNA clone encoding GC-B was isolated by low-stringency screening of a rat brain cDNA library using GC-A cDNA as a probe. The deduced amino acid sequence of GC-B is 78% identical with GC-A within the intracellular region, but 43% identical within the extracellular domain. Cyclic GMP concentrations in cells transfected with GC-A were half-maximally elevated at 3 nM ANP, 25 nM brain natriuretic peptide (BNP), and 65 nM atriopeptin 1, while 25 microM ANP, 6 microM BNP, and greater than 100 microM atriopeptin 1 were required for half-maximal stimulation of GC-B. The potencies of natriuretic peptides on GC-A and GC-B activity are therefore markedly different; furthermore, despite the specificity of GC-B for BNP, the relatively high BNP concentration required to elicit a response suggests the possible presence of a more potent, unidentified natural ligand.