Natriuretic peptide receptor-B (NPRB) was visualized in rat tissues by in vitro autoradiography, using its putative physiological agonist C-type natriuretic peptide (CNP). In initial studies, we determined that atrial natriuretic peptide (ANP) is not a suitable ligand for labeling the NPRB: in tissues reported to contain NPRB transcripts, CNP did not inhibit [125I]ANP binding except to NPRC sites. Therefore, to visualize the NPRB we used 125I[Tyr(o)]-CNP as a radioligand with an excess of NPRC-blocking peptide: C-ANP. With this approach we detected the highest number of NPRB-like sites in the pars intermedia of the pituitary gland. A large number of these sites were present in pituitary neural and anterior lobes, area postrema, adrenal medulla and cortex. A moderate NPRB population was observed in the subfornical organ, plexiform layer of the olfactory bulb and kidney. Low concentrations of NPRB were noted in the cerebellum and cerebrum but not in the choroid plexus and pia-arachnoid. Saturation experiments performed on cerebellum sections revealed a very low concentration (Bmax 4.8 fmol/mg protein) of high affinity (Kd 1.2 nM) NPRB-like sites. This study is the first demonstration of 125I[Tyr(o)]-CNP binding sites with characteristics of the NPRB in intact tissues.