Abstract Mixed cultures of Saccharomyces cerevisiae CBS 8066 and Kluyveromyces fragilis were used for continuous biomass production on media containing whey permeate and glucose (WG) or whey permeate, maltose and glucose (WGM). Glucose and maltose were provided from a totally or a partially hydrolysed starch. For each medium the effects of two yeast extract concentrations (0.1 and 0.5 g/litre) were investigated. The yeast extract concentration markedly affected the equilibrium of the co-culture and the cell yield which was also influenced by the dilution rate. The yield varied from 0.26 to 0.48 g/g. The results obtained were due to the different kinetics of glucose transport in Kluyveromyces fragilis and Saccharomyces cerevisiae. Kluyveromyces fragilis possessed two glucose carriers, present at all the tested dilution rates, characterised by their affinity constants which were much higher than those present in Saccharomyces cerevisiae.