The green microalga Haematococcuspluvialis was cultured with different concentrations of NaNO3 to determine the effect on cell growth and astaxanthin accumulation. The optimum nitrate concentration to obtain astaxanthin and to avoid the cessation of cell division was 0.15 g/l NaNO3. The ratio chlorophyll a/total carotenoids proved a good physiological indicator of nitrogen deficiency in the cell. The effect of different carbon sources, malonate and acetate, on astaxanthin accumulation was also studied; up to 13 times more carotenoids per cell were accumulated in cultures with malonate than in cultures without this compound. The pigment analysis was performed by a new low toxicity HPLC method capable of separating chlorophylls a and b, carotenes and xanthophylls in a short-period of time, using low volumes of solvents and with an economical price. With this method even echinenone was separated, which had been unsuccessful by any other method. (C) 2004 Elsevier Ltd. All rights reserved.