Interfacial activation via surfactant (Tween 80, Triton X-100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross-linked enzyme aggregates (CLEA (R)) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non-pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899-904, 2016