Alkyl glycosides are interesting intermediates for the production of biodegradable surfactants. Synthesis of ethyl beta-D-fructofuranoside by invertase-catalysed ethanolysis of sucrose has been extensively reported in literature. However, this procedure yields mixtures of glucose, fructose, sucrose and ethyl beta-D-fructofuranoside. Purification of ethyl beta-D-fructofuranoside from such mixtures by chromatographic methods is laborious, difficult to scale up and requires organic solvents. The yeast Hansenula polymorpha grows rapidly on glucose, fructose and sucrose. Sucrose hydrolysis in this yeast is catalysed by an intracellular alpha-glucosidase ('maltase'); consequently, H. polymorpha should be unable to hydrolyse ethyl beta-D-fructofuranoside. Indeed, aerobic cultivation of H. polymorpha on sugar mixtures obtained by invertase-catalysed ethanolysis of sucrose resulted in the complete removal of contaminating sugars, leaving ethyl beta-D-fructofuranoside as the sole organic compound in culture supernatants. Pure ethyl beta-D-fructofuranoside was recovered from the supernatants by mixed-bed ion exchange chromatography with an 86% yield.