Guanylyl cyclase-B receptor (GC-B) is a membrane receptor that induces intracellular accumulation of cGMP when a specific ligand, C-type natriuretic peptide (CNP), binds to the extracellular ligand-binding domain (ECD). Despite of its medical and biological importance, characterization of GC-B is hampered by limited amounts of protein obtainable. To circumvent this problem, a method was developed for rapidly and semi-automatically establishing stable cell lines specialized for large-scale production. This method, utilizing a bicistronic expression vector for co-expressing a green fluorescent protein and FACS-based selection of high-expressing cells, is generally applicable. It worked particularly well with the ECD and yielded highly purified ECD at 1 mg/l of culture medium by affinity chromatography using modified CNPs. Measurements of ligand-binding and guanylyl cyclase activities for various natriuretic peptides showed that, as expected, CNP is by far the most potent agonist of GC-B with IC50 of similar to 7.5 nM. This value is at least an order of magnitude larger than that reported earlier but similar to that established with the guanylyl cyclase-A receptor for its ligand, atrial natriuretic peptide. The methods developed here will be useful, at the least, for characterizing other members of the guanylyl cyclase receptor family. (C) 2012 Elsevier Inc. All rights reserved.