The yeast Candida rugosa produces multiple extracellular lipases. The production of extra- and intracellular lipases was investigated in continuous cultures using a sole or different mixtures of carbon sources. Also, the effect of different C:N ratios was tested. Lipase productivity in continuous cultures increased by 50% compared with data obtained from batch fermentations and depended on the dilution rate applied. Maximum yields relative to consumed substrate were obtained with oleic acid at low dilution rate. It was found that during nitrogen limitation, lipase activity was suppressed. All carbon source mixtures tested allowed both cell growth and lipase production, but extra- and intracellular lipase activities were affected by the combination of substrates used. Maximum extracellular lipolytic productivity was attained with lactic and oleic acid mixtures, probably due to the non-repressor effect of these carbon sources. The chemical composition of the biomass also depended on the type of substrate used and was related to the accumulation of lipidic compounds as intracellular inclusions, which were observed when oleic acid was used as the carbon source. The results obtained were compared with previous data from batch and fed-batch cultures in order to select the best process strategies for the lipase production with C rugosa. The best lipase yields were obtained in fed-batch fermentations using oleic acid. (C) 2003 Society of Chemical Industry.