Atrial natriuretic factor (ANF), a 28-amino-acid peptide secreted from the mammalian heart, is known to be cleared rapidly from the circulation. In vitro and in vivo studies implicate the kidney as an important site for clearance and subsequent degradation of atrial natriuretic factor. We have observed that atrial natriuretic factor is inactivated rapidly by rabbit kidney brush-border membranes. The rate of degradation of ANF measured by the loss of bioactivity followed a similar time-course to the decrease in peptide peak area measured by high-performance liquid chromatography. Interestingly, inactivation of ANF produced only a single major degradation product, which was isolated and purified. Sequence analysis revealed that the product had the same sequence of amino acids as ANF with the Cys-7-Phe-8 bond cleaved and the disulfide bridge between Cys-7 and Cys-23 remaining intact. As the renal brush border contains an abundance of proteolytic activities, it is surprising that this peptide is cleaved primarily at a single peptide bond.