Biodiesel is usually produced by the transesterification of vegetable oils and animal fats with methanol, catalyzed by strong acids or bases. This study introduces a novel biodiesel production method that features direct base-catalyzed methanolysis of the cellular biomass of oleaginous yeast Rhodosporidium toruloides Y4. NaOH was used as catalyst for transesterification reactions and the variables affecting the esterification level including catalyst concentration, reaction temperature, reaction time, solvent loading (methanol) and moisture content were investigated using the oleaginous yeast biomass. The most suitable pretreatment condition was found to be 4 g L-1 NaOH and 1: 20 (w/v) dried biomass to methanol ratio for 10 h at 50 degrees C and under ambient pressure. Under these conditions, the fatty acid methyl ester (FAME) yield was 97.7%. Therefore, the novel method of direct base-catalyzed methanolysis of R. toruloides is a much simpler, less tedious and time-consuming, process than the conventional processes with higher FAME (biodiesel) conversion yield. (C) 2014 Elsevier Ltd. All rights reserved.