In the present paper we report the structural characterization of two genetic mutants of human serum albumin: albumin Vanves, a very rare, electrophoretically fast variant of French origin, and albumin Verona, a slow-migrating variant which is the most frequently observed in Italy and which possesses the same electrophoretic mobility as albumin B. Both variants were isolated from the sera of healthy heterozygous subjects. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585) in both cases. The modified fragments were then isolated on a preparative scale by HPLC and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by HPLC, established the mutation responsible for albumin Vanves as 574 Lys----Asn and the molecular defect of albumin Verona as 570 Glu----Lys, both probably due to point mutations in the structural genes. The amino-acid substitutions found in albumins Verona and Vanves are consistent with the electrophoretic mobilities observed for the native proteins at pH 8.6.