The goal of this study was to test the ability of small high density lipoproteins (small HDL) to bind human alpha-atrial natriuretic peptide (alpha-hANP), an amyloidogenic peptide whose involvement in cardiac pathologies is gaining increasing clinical evidence. After incubation of HDL with labeled ANP, the peptide associated to lipoprotein was detectable only in small HDL containing preparations. HDL-associated alpha-[(125)I]hANP was subjected to chromatography, electrophoresis, and autoradiography. The autoradiograph showed two radioactive bands, whose molecular weight was consistent with the chromatographic pattern. Immunoblotting showed the presence of apo A-I in both autoradiographic bands. The proteins of the main band were electroeluted, incubated with labeled ANP, and subjected to two-dimensional electrophoresis followed by autoradiography. The mass spectrometry and molecular weight analyses of the radioactive spot demonstrated the presence of an apo A-I dimer. This finding provided a novel solid evidence that small HDL via apo A-I dimer are involved in the ANP sequestration and thus may play a role in preventing amyloid fibril formation.