Atriopeptin (AP) 24, containing amino acids Ser103-Tyr126 of the carboxy-terminal portion of the atrial natriuretic peptide prohormone, was degraded rapidly by rabbit kidney brush border membranes. The rate of degradation of AP24 measured by the loss of vasorelaxant activity followed a similar time course to the decrease in peptide peak area measured by high-performance liquid chromatography. Inactivation of AP24 produced peptide fragments which were separated by HPLC. The major products were purified individually and their peptide sequences determined. Results indicate that AP24 was proteolytically cleaved at three peptide bonds: Ser103-Ser104, Cys105-Phe106 and Ser123-Phe124. des-Ser103-AP24 had similar vasorelaxant activity to AP24, while AP24 cleaved at Cys105-Phe106 was inactive. Regarding the proteolytic cleavage at Ser123-Phe124, there was an accumulation of the C-terminal tripeptide, Phe-Arg-Tyr, only at the later time points of the incubation. Degradation experiments were repeated with an amino- and carboxy-terminal protected peptide, acetyl-AP24-amide. Peptide sequence analysis of the major degradation products of this peptide revealed that the critical peptide bond cleaved was Cys105-Phe106. We conclude that the Cys-Phe peptide bond renders atrial peptides highly susceptible to proteolysis by renal brush border membranes, resulting in inactivation.