The feasibility of using intermittent cycle extended aeration system (ICEAS) sludge as a lipid feedstock for biodiesel production was investigated. The main effects of in situ transesterification parameters, reaction temperature (30-70 degrees C), reaction time (4-24 h), catalyst concentration (1-5% v/v), and proportion of methanol to dry sludge (5-25 ml/g) at five-levels as well as their simultaneous interactions were evaluated to develop an empirical model. Optimized conditions were obtained at 60 degrees C, 4.65% (v/v) H2SO4, 17.84 h reaction time, and 5:1 methanol to dry sludge proportion (ml/g), leading to a maximum of 18.58% (w/w) biodiesel yield with 94.23% fatty acid methyl ester content. This result was higher in comparison with yields derived from conventional activated sludge, membrane bioreactor and anaerobic-anoxic-oxic processes. The ICEAS technology advantages are owned to its different configurations leading to production of one blended sludge, shorter hydraulic retention time and higher chemical oxygen demand to nitrogen ratios. The predominance of fatty acid methyl esters such as pal-mitic, oleic, palmitoleic, stearic, linoleic and myristic acid methyl ester, in the obtained biodiesel, indicated suitability of ICEAS sludge as feedstock for biodiesel production. Published by Elsevier Ltd.