AbstractThe physiology of Hanseniaspora uvarum K5 was studied in glucose‐limited chemostat cultures and upon glucose pulse. Up to a dilution rate of 0·28 h−1, glucose was completely metabolized in biomass and CO2. Above this value, increase in the dilution rate was accompanied by sequential production of metabolites (glycerol, acetate and ethanol) and decrease in cell yield. Similar results were observed upon glucose pulse. From the enzyme activities (pyruvate dehydrogenase, pyruvate decarboxylase, NAD and NADP‐dependent acetaldehyde dehydrogenases, acetyl coenzyme A synthetase and alcohol dehydrogenase) and substrate affinities, the following conclusions were drawn with respect to product formation of cells: (1) pyruvate was preferentially metabolized via pyruvate dehydrogenase, when biomass and CO2 were the only products formed; (2) acetaldehyde formed by pyruvate decarboxylase was preferentially oxidized in acetate by NADP‐dependent aldehyde dehydrogenase; acetate accumulation results from insufficient activity of acetyl‐CoA synthetase required for the complete oxidation of acetate; (3) acetaldehyde was oxidized in ethanol by alcohol dehydrogenase, in addition to acetate production.