The synthesis of biodiesel with free lipases starting from refined rapeseed oil and low quality cooking oils was evaluated. A two-enzyme-one-pot system was developed, which gave ethyl ester yields of 96% with less than 1% of residual fatty acids before purification. In the first reaction step Thermomyces lanuginosus lipase was employed, which exhibited high activity of 600 nmol/s/mg on triglycerides. Triglyceride turnover was nearly 100% and ethyl ester-glyceride mixtures with 3-4% of fatty acids accumulated. In the second reaction step, Pseudozyma antarctica lipase B dissolved in deep eutectic solvents was applied successfully for residual glyceride transesterification and fatty acid esterification. After phase separation, biodiesel was purified in a one-step refinement to yield a product with 97.6% ethyl ester content. Glycerol of high quality was obtained in a single distillation step from the hydrophilic deep eutectic solvent phase. Comparative trials with several used cooking oils revealed, that.the first transesterification step worked equally well, while reaction velocity of the second step was lower than with refined oils. More than 80% ethyl ester yield were obtained with all used cooking oils tested. (C) 2015 Elsevier Ltd. All rights reserved.