Previous purification of a crude extracellular enzyme preparation from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular masses (58 and 62 kDa, respectively). These enzymes were purified but the lipases were forming active aggregates with a molecular mass higher than 200 kDa. In this work we developed a purification method following three steps: ammonium sulfate precipitation, sodium cholate treatment and ethanol/ether precipitation, and anion exchange chromatography which allowed the sequential disaggregation of the isoenzymes. Pure and monomeric Lip2 and Lip3 were characterized according to pI, glycosylation and activity for p-nitrophenol esters and triacylglycerols of varying acyl chain. Lip3 was the best catalyst for the hydrolysis of the simple esters and triacylglycerols with short and medium acyl chains.