BACKGROUND Measurement of brain natriuretic peptide (BNP) in plasma is useful for the diagnosis and prognosis of heart failure. However, the molecular forms of BNP, especially proBNP in the blood, have been poorly characterized. METHODS To investigate the forms of proBNP in blood, antibodies against four proBNP synthetic peptides (1-13, 22-27, 28-40, and 47-54) were developed and characterized for their reactivity to proBNP by ELISA. Using the antibodies and a monoclonal antibody specific to the carboxyl terminal histidine of BNP, a radioimmunoassay for proBNP was constructed. BNPs in plasma from heart failure patients were analyzed by gel-filtration HPLC followed by immunoassay for BNP and proBNP. RESULTS Two of four antibodies against proBNP synthetic peptides reacted with both r-proBNP and BNP in plasma from the patients. Gel-filtration HPLC analysis showed two major peaks of immunoreactive BNP (high MW BNP (36 kDa) and low MW BNP (4 kDa)). The immunoassay demonstrated that almost the full-length of proBNP was contained in the high MW BNP fractions. CONCLUSIONS ProBNP was more stable than its mature form of BNP in circulation after secretion, and it suggested the usefulness of proBNP measurement.