A novel natriuretic peptide receptor, which we have termed natriuretic peptide receptor D (NPR-D), has been cloned and characterized. cDNAs related to the natriuretic peptide receptor (NPR) were amplified by PCR from a template of poly(A)-rich RNA isolated from the eel gill. Sequencing of the PCR products revealed the presence of a new clone that showed about 70% sequence identity to the eel type-C receptor, NPR-C. The PCR fragment was used to determine the tissue distribution of the new NPR-D message by an RNase protection assay, which gave the strongest signal in brain samples, and then used to screen a brain library to obtain a full-length cDNA clone. The cDNA clone predicted a protein of 500 amino acids containing a signal sequence and a hydrophobic transmembrane segment. The predicted sequence also contained the NPR motif which is essential for the binding of natriuretic peptides. The protein NPR-D was expressed in COS cells and shown to have high affinities for eel and rat natriuretic peptides. The newly cloned NPR-D has a short cytoplasmic tail; in this respect, NPR-C and NPR-D are very similar and form a subfamily of the NPR family. Affinity labeling indicated that NPR-D exists as a disulfide-linked tetramer. This is a marked contrast to the homodimeric structure of NPR-C. HS-142-1, a non-peptide natriuretic peptide receptor antagonist of microbial origin previously shown to be selective for the guanylate-cyclase-coupled receptors NPR-A and NPR-B, competitively inhibited the binding of "'I-labeled eel natriuretic peptide to eel NPR-D, whereas it did not affect the binding activity of eel NPR-C, suggesting that HS-142-1 is an antagonist that recognizes the tetrameric structures of NPR since the guanylate-cyclase-coupled receptors have also been demonstrated to exist as tetramers.Keywords: antagonist; cloning; eel ; natriuretic peptide; receptor.The natriuretic peptide system is involved in the central and peripheral control of body fluid volume and blood pressure [ 1 -61. In mammals, three peptides, natriuretic peptides A, B and C (ANP, BNP and CNP, respectively), have been identified as the members of the natriuretic peptide family and shown to have diverse biological actions including diuresis, natriuresis, vasorelaxation, anti-mitogenesis, and modulation of other regulatory systems. Comparative studies using non-mammalian species revealed the presence of additional members; for example, two types of CNP have been cloned from the frog [ 7 ] , and from the eel ventricle, ventricular natriuretic peptide has been isolated and suggested to be a fourth member of the family [8].