We have established a specific radioimmunoassay (RIA) for C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, and have elucidated its tissue distribution and molecular form. In rats, high concentrations of CNP-like immunoreactivity (-LI) were detected in the anterior lobe (19.8 +/- 8.6 pmol/g) and neurointermediate lobe (4.64 +/- 0.74 pmol/g) of the pituitary gland. CNP-LI was present throughout the brain with its high concentrations in the hypothalamus and cerebellum. Small amounts of CNP-LI were also detected in the lower part of gastrointestinal tract and the kidney. However, no significant amount of CNP-LI was present in other organs including the heart. Considerable amounts of CNP-LI were detected throughout the human brain. High performance-gel permeation chromatography coupled with the RIA detected two peaks of CNP-LI in the rat brain; CNP and presumably its N-terminally elongated form with 53 amino-acid residues, CNP-53. These findings indicate that the tissue distribution and processing pattern of CNP are clearly different from those of atrial natriuretic peptide and brain natriuretic peptide and suggest possible roles of CNP as a neurotransmitter or neuromodulator rather than as a cardiac hormone.