A gradient reversed-phase HPLC method was developed for the separation of astaxanthin esters and the isomers of astaxanthin from the unsaponified and saponified pigment extracts of the microalga Haematococcus pluvialis. Four kinds of isomers of astaxanthin and astaxanthin esters, (3S,3‘S)-trans-astaxanthin, (3S,3‘S)-9-cis-astaxanthin, (3S,3‘S)-13-cis-astaxanthin, (3R,3‘R)-trans-astaxanthin, and their esters, were separated and identified. A small amount of (3S,3‘S)-15-cis-astaxanthin was also detected from the saponified extract. In addition, a chromatographic purification method was established for the preparation of natural trans-astaxanthin from the saponified extract of H. pluvialis. With this method, 3.7 mg of astaxanthin was isolated from 1 g of dry biomass of H. pluvialis. The purified astaxanthin contained approximately 97.7% trans-astaxanthin, 1.1% cis-astaxanthin, and 1.2% impurity. Keywords: Astaxanthin; astaxanthin esters; purification; carotenoids; Haematococcus pluvialis; HPLC