Biodiesel from microalgae seems to be the only renewable biofuel that has the potential to completely replace the petroleum-derived transport fuels. Therefore, improving lipid content of microalgal strains could be a cost-effective second generation feedstock for biodiesel production. Lipid accumulation in Scenedesmus obliquus was studied under various culture conditions. The most significant increase in lipid reached 43% of dry cell weight (dcw), which was recorded under N-deficiency (against 12.7% under control condition). Under P-deficiency and thiosulphate supplementation the lipid content also increased up to 30% (dcw). Application of response surface methodology in combination with central composite rotary design (CCRD) resulted in a lipid yield of 61.3% (against 58.3% obtained experimentally) at 0.04, 0.03, and 1.0 g l(-1) of nitrate, phosphate, and sodium thiosulphate, respectively for time culture of 8 days. Scenedesmus cells pre-grown in glucose (1.5%)-supplemented N 11 medium when subjected to the above optimized condition, the lipid accumulation was boosted up to 2.16 g l(-1), the value similar to 40-fold higher with respect to the control condition. The presence of palmitate and oleate as the major constituents makes S. obliquus biomass a suitable feedstock for biodiesel production.