Biodiesel as alkyl esters derived from vegetable oils has considerable advantages in terms of environmental protection. In the present work, methyl esters were produced from sunflower oil by lipase-catalyzed reactions. The effect of organic solvent on alcoholysis rate was investigated and highest conversion (80%) was found in n-hexane and petroleum ether. Among several microbial lipases tested for alcoholysis activity in a solvent-free system, the best conversion (>90%) was found with lipase from Pseudomonas fluorescens (Amano AK) at the highest molar equivalent of oil:methanol (1:4.5). To reduce inactivation of commercial immobilized lipases by methanol, a three-step protocol consisting of the stepwise addition of I M equivalent of methanol at 5 h intervals, was developed. Also with immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) high conversion (>80%) was possible. Moreover, the transesterification could be conducted for at least 120 h during five batch runs without significant loss of activity. (C) 2003 Elsevier Science Inc. All rights reserved.