Microbial lipid is a potential alternative feedstock for the biodiesel industry. New culture strategies remain to be developed to improve the economics of microbial lipid technology. This work describes lipid production by the oleaginous yeast Rhodosporidium toruloides Y4 using a 15-l bioreactor with different substrate feeding strategies. Among these strategies, the intermittent feeding mode gave a lipid productivity of 0.36 g l(-1) h(-1), whereas the constant glucose concentration II (CC-II) mode gave the highest lipid productivity of 0.57 g l(-1) h(-1). The repeated fed-batch mode according to the CC-II mode was performed with a duration time of 358 h, and the overall lipid productivity was 0.55 g l(-1) h(-1). Our results suggested that substrate feeding modes had a great impact on lipid productivity and that the repeated fed-batch process was the most appealing method by which to enhance microbial lipid production.