We have investigated the role and the kinetic properties of the Hxt5 glucose transporter of Saccharomyces cerevisiae. The HXT5 gene was not expressed during growth of the yeast cells in rich medium with glucose or raffinose. However, it became strongly induced during nitrogen or carbon starvation. We have constructed yeast strains constitutively expressing only Hxt5, Hxt1 (low affinity) or Hxt7 (high affinity), but no other glucose transporters. Aerobic fed-batch cultures at quasi steady-state conditions, and aerobic and anaerobic chemostat cultures at steady-state conditions of these strains were used for estimation of the kinetic properties of the individual transporters under in vivo conditions, by investigating the dynamic responses of the strains to changes in extracellular glucose concentration. The K(m) value and the growth properties of the HXT5 single expression strain indicate that Hxt5 is a transporter with intermediate affinity.