-We investigated the molecular mechanism underlying the processing of pro-B-type natriuretic peptide (proBNP). Rat neonatal atrial and ventricular myocytes were cultured separately. We examined the molecular forms of secreted and intracellular BNP in atrial and ventricular myocytes; levels of corin and furin mRNA in atrial and ventricular myocytes; the effect their knockdown on proBNP processing; plasma molecular forms of BNP from rats and humans with and without heart failure; and the impact of the distance between the glycosylation and cleavage sites in wild-type and mutant human proBNP, expressed in rat myocytes transfected with lentiviral vectors. BNP was the major molecular form secreted by atrial and ventricular myocytes. Transfection of furin siRNA reduced proBNP processing in both atrial and ventricular myocytes; however, transfection of corin siRNA did not reduce it. BNP was the major molecular form in rat plasma, whereas proBNP was the major form in human plasma. The relative fraction of human BNP in rat myocytes expressing human proBNP was about 60%, but increasing the distance between the glycosylation and cleavage sites through mutation, increased the processed fraction correspondingly. These results suggest that proBNP is processed into BNP intracellularly by furin. The level of proBNP processing is lower in humans than rats, most likely due to the smaller distance between the O-glycosylation and cleavage sites in humans.B-type natriuretic peptide; atrial natriuretic peptide; furin; processing; corin B-TYPE (ALSO KNOWN AS BRAIN) NATRIURETIC PEPTIDE (BNP) is a cardiac hormone mainly expressed in the ventricles (14). Ventricular expression of BNP increases in response to pressure overload, volume overload, and ischemic injury, and plasma BNP levels increase in proportion to disease severity in patients with heart failure, myocardial infarction, pulmonary hypertension, or cardiac hypertrophy (13). Whereas tissue concentrations of BNP are much higher in the atria than the ventricles (1), and BNP secreted from the atria is also important in cases of atrial fibrillation and atrial overload (6). The precursor proBNP is believed to be cleaved to BNP and NH 2 -terminal proBNP, as it is secreted from myocytes (13), and both molecules have been used for more than a decade as biochemical markers of heart failure. However, several recent studies have shown that in addition to BNP and NH 2 -terminal proBNP, levels of uncleaved proBNP are also greatly increased in heart failure (15,23,28).The precise mechanism by which proBNP is processed is not fully understood at present. Specifically, the molecular form of BNP within cells, the mechanism of proBNP processing, the enzymes catalyzing proBNP processing, whether the patterns of BNP secretion differ between the atria and ventricles, and whether the pattern of BNP secretion differs from that of atrial natriuretic peptide (ANP) all remain poorly understood. Interestingly, recombinant proBNP derived from mammalian cells has seven sites of O-linked oligosaccharide atta...