The present paper investigates the comparative enzymatic hydrolysis of cod liver oil with Candida cylindracea lipase (CCL) and Candida rugosa lipase (CRL) in biphasic solvent system for the production of free fatty acids (FFAs). These two lipases were characterized for their physiochemical properties with methods such as BET surface area analyzer, particle size analyzer and Fourier transform infrared (FT-IR) spectroscopy to find the relationship between the characteristic and activity. The yield of FFAs from cod liver oil was studied by varying suitable reaction conditions such as pH, temperature, agitation speed, water and solvent concentrations. The maximum FFAs yields of 36.3 and 26.1 wt% FFAs (equivalent to 122.9 and 88.6 mu moles FFAs formed/ml) were obtained for CCL and CRL respectively under the most suitable reaction conditions. The Michaelis-Menten kinetic model was found to fit satisfactorily for the hydrolysis of cod liver oil with both CCL and CRL enzymes and the kinetic parameters such as maximum rate of reaction (V-max) and rate constant (K-M) were calculated. The activation energy (E = 21.3 kJ/mol) demonstrates that the hydrolysis of cod liver oil is kinetically controlled and free from mass transfer limitations under the experimental conditions studied. (C) 2012 Elsevier B. V. All rights reserved.