High performance gel permeation chromatography (HP-GPC) followed by four radioimmunoassays (RIAs) devised to amino acids (a.a.) 1-30, 31-67, 79-98, and 99-126 of the 126 a.a. atrial natriuretic factor (ANF) prohormone revealed that the proANF(1-30) assay immunoreactivity in plasma is 50% proANF(1-30) and 50% proANF(1-98). The HP-GPC evaluation of plasma followed by proANF(31-67) and ANF [i.e., proANF(99-126)] assays revealed that proANF(31-67) and ANF circulate as distinct peptides. the HP-GPC plasma examination followed by proANF(79-98) assay immunologically recognized three peaks in plasma consistent with proANF(1-98), -(68-98), and -(79-98). Similar HP-GPC evaluation of urine followed by these RIAs indicated that the proANF(1-30), -(79-98), and ANF assays only recognize 500 mol.wt. or less peptides, and the proANF(31-67) RIA recognizes a nearly intact proANF(31-67) with only two to three amino acids removed during processing of this peptide.