The alcoholysis (transesterification) of the refined cotton seed oil of Turkish origin with primary and secondary alcohols was investigated in the presence of an immobilized enzyme from Candida antarctica, commercially called Novozym 435 in a solvent-free medium. The optimum conditions of the methanolysis were as follows: 30% enzyme based on oil weight; oil/alcohol molar ratio 1:4; temperature: 50 °C and reaction time: 7 h. Maximum methyl esters (ME) yield was 91.5%. At the same conditions cotton seed oil was converted with short-chain primary and secondary alcohols to its corresponding esters with conversions between 72% and 94%. Our results indicated that alcoholysis products of cotton seed oil could be used as valuable intermediates in oleochemistry.