The fast growing unicellular green microalga Chlorella zofingiensis has attracted interest as a promising. organism for commercial production of the high-value carotenoid astaxanthin [Del Campo JA, Rodriguez H, Moreno J, Vargas MA, Rivas J, Guerrero MG. Accumulation of astaxanthin and lutein in Chlorella zofingiensis. Appl Microbiol Biotechnol 2004;64:848-54; Ip PR Wong KH, Chen F. Enhanced production of astaxanthin by the green microalga Chlorella zofingiensis in mixotrophic culture. Process Biochem 2004;39:1761-6; Ip PF, Chen, F. Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark. Process Biochem 2005;40:733-8.]. In this study, the reactive nitrogen species (RNS) peroxynitrite and the reactive nitrogen intermediate (RNI) nitryl chloride were applied to induce carotenogenesis leading to enhanced astaxanthin formation in C. zofingiensis in dark heterotrophic culture. Both the RNS and RNI when applied at an appropriate concentration stimulated astaxanthin biosynthesis in C. zofingiensis. Cultures treated with 1 mM peroxynitrite led to an enhanced yield of astaxanthin from 9.9 to 11.78 mg 1(-1). Nitryl chloride generated from the reaction mixture of 0.1 mM sodium nitrite with 0.5 mM sodium bypochlorite also increased the astaxanthin yield to 10.99 mg 1(-1). The present study is the first report demonstrating the possibility of using RNS and RNI for the enhancement of astaxanthin production in heterotrophic microalgal culture. (c) 2005 Elsevier Ltd. All rights reserved.