Dark-heterotrophic growth of microalgae eliminates the requirement for light and therefore, facilitates large-scale cultivation of microalgae, especially in denser cultures [Trends Biotechnol. 14 (1996) 421]. In this study, the heterotrophic production potential of the secondary carotenoid astaxanthin by the green microalga Chlorella zofingiensis was investigated. The alga showed excellent growth on glucose-supplemented media in batch culture. The maximum specific growth rate and astaxanthin yield were 0.031 h(-1) and 10.3 mg l(-1), respectively obtained at glucose concentrations of 20 and 50 g l(-1), which were the highest ever reported in heterotrophic algal cultures. In the absence of light, formation of secondary carotenoids was mostly dependent on the initial carbon and nitrogen balance in the medium; enhanced biosynthesis of secondary carotenoids including astaxanthin was found in the medium with a high C/N ratio (i.e. C/N ratio = 180). The light-independent astaxanthin-producing ability of C zofingiensis suggested that the alga might be potentially employed for commercial production of astaxanthin on a large-scale. (C) 2004 Elsevier Ltd. All rights reserved.