The fast growing unicellular green microalgae Chlorella protothecoides has attracted interest as a promising organism for commercial production of a high-value carotenoid, lutein, by heterotrophic fermentation. Effects of two oxidant-forming reactive oxygen species (ROS) on the biomass concentration, and yield and content of lutein in batch culture of heterotrophic Chlorella protothecoides were investigated in this study. The addition of 0.1 mmol/L H(2)O(2) and 0.01 mmol/L NaClO plus 0.5 mmol/L Fe(2+) to the culture led to the generation of (.)OH and enhanced the lutein content from 1.75 to 1.90 and 1.95 mg/g, respectively. The lutein content further increased to 1.98 mg/g when 0.01 mmol/L H(2)O(2) and 0.5 mmol/L NaClO were added to generate (1)O(2). The maximum yield of lutein (28.5, 29.8 and 31.4 mg/L) and a high biomass concentration (15.0, 15.3 and 15.9 g/L) were also achieved through the above treatments. The results indicated that 1O2 could promote lutein formation and enhance lutein production in heterotrophic Chlorella protothecoides. Moreover, (1)O(2) produced from the reaction of H(2)O(2) and NaClO was more effective in enhancing lutein production and reducing biomass loss than (.)OH from the reaction of H(2)O(2) or NaClO plus Fe(2+).