Sophorolipids. obtained by a two-stage process starting from deproteinized whey concentrate using Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214, were compared to products from one-stage processes, using different lipidic compounds as substrates. Results showed that above all carbon source and not cultivation conditions had a distinct influence on the composition of the crude product mixture and therefore on the physicochemical and biological properties of the sophorolipids, such as, for example, surface activity, cytotoxicity and stability against hydrolases. The results were completed by corresponding data for purified mono- and diacetylated (17-hydroxyoctadecenoic)-1＇,4 "-lactonized sophorolipids. Crude sophorolipid mixtures showed moderate to good surface active properties (SFTmin 39 mN m(-1), CMC 130 mg l(-1)), water solubilities (2-3 g l(-1)) and low cytotoxicities (LC50 300-700 mg l(-1)). In contrast, purified sophorolipids were more surface active (SFTmin 36 mN m(-1), CMC 10 mg l(-1)), less water soluble (max. 70 mg l(-1)) and showed stronger cytotoxic effects (LC50 15 mg l(-1)). Incubation of crude sophorolipid mixtures with different hydrolases demonstrated that treatment with commercially available lipases such as from Candida rugosa and Mucor miehei distinctly reduced the surface active properties of the sophorolipids, while treatment with porcine liver esterase and glycosidases had no effect.