Fatty acid ethyl esters (FAEEs) were produced from waste cooking oil (WCO) using the immobilized Novozym 435 (Novozymes) lipase in a solvent-free system. WCO contained high molar mass degradation compounds that did not affect the lipase activity but remained as a minor component of the resulting FAEEs. The enzyme-catalyzed FAEE production was investigated using a central composite design. The best experimental conditions were obtained with an enzyme loading of 5 wt % and an ethanol/oil molar ratio of 61. Under these conditions and at the optimal enzyme temperature of 40 degrees C, FAEE mixtures with a total ester content of 77.2 and 89.5 wt % were produced after 10 and 24 h, respectively. Treatment of the spent biocatalyst with tert-butanol could restore the transesterification activity of the biocatalyst to its original value.