A new procedure was developed for isolation of two major serum amyloid A (SAA) isotypes SAA1 and SAA2 from acute-phase plasma of mice. The procedure included preparation of high-density lipoproteins (HDLs) and their separation by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The SAA proteins (Mr 12,000) were electroeluted and afterwards purified from SDS by gel permeation chromatography on a Fractogel TSK-40F column in aqueous 50% acetonitrile-0.1% TFA. Finally, the SAA proteins free from SDS were fractionated by high-performance liquid chromatography on a Vydac 214TP54 column (250 x 4.6 mm I.D., particle size 5 microm), yielding two major fractions with k=5.2 and k=5.5. The N- and C-terminal sequence analyses and mass spectrometry demonstrated the purity of these two major fractions and their identity with apo SAA1 (k=5.2) and apo SAA2 (k=5.5). The developed procedure is applicable to small amounts of pooled murine plasma (6-7 ml) and could be readily modified from small to large scale preparations.