To develop a radioimmunoassay (RIA) specific for human C-type natriuretic peptide (hCNP), we used a highly specific antiserum raised in rabbits. Quantitative inhibition tests with various natriuretic peptides demonstrated that the 50% inhibitory dose of hCNP was 15 fmol, whereas those of other natriuretic peptides were 10(5)-fold higher, indicating a specificity satisfactory for determining concentrations of hCNP in tissues. Using this antiserum, we detected immunoreactive hCNP (ir-hCNP) in various regions of human brain and spinal cord, as well as in cerebrospinal fluid (CSF). The ir-hCNP concentrations in human neural tissues were approximately 10-fold higher than those of immunoreactive human atrial natriuretic peptide (ir-hANP). The mean (+/- SD) concentration of ir-hCNP (72.0 +/- 17.8 ng/L) in CSF also was 10-fold higher than that of ir-hANP (5.2 +/- 2.1 ng/L). Using gel-permeation chromatography, we identified two molecular forms of ir-hCNP in brain and CSF: a 2-kDa form corresponding to mature hCNP, which is composed of 22 amino acid residues (hCNP-22), and a 5- to 6-kDa form corresponding to an N-terminally extended molecule (hCNP-53). The latter form was predominant in brain; the former was the main constituent of hCNP in CSF. These results support the hypothesis that hCNP is a major natriuretic peptide, is synthesized in human brain, and functions in human central nervous tissues.