Glucose transport in the yeast Kluyveromyces marxianus proceeds by two functionally and presumably structurally distinct transporters depending on the carbon source of the culture medium. In lactose-grown cells, glucose was taken up through a high-affinity H+-sugar symporter (Km = 0.09 mM), whereas a low-affinity transporter (Km = 3.5 mM) was utilized in glucose-grown cells. The two transporters exhibited different substrate specificities. Galactose was demonstrated to be a selective substrate of the H+-glucose symporter (Km = 0.14 mM) and did not significantly enter glucose-grown cells. Fructose was a preferential substrate of the low-affinity carrier (Km = 3.5 mM), but it entered lactose-grown cells through a high-affinity H+-fructose symporter distinct from the H+-glucose one. Other putative substrates of the two glucose transporters were identified by competition experiments. 2-Deoxyglucose recognized both carriers with a similar affinity, while the non-phosphorylatable analogues 6-deoxyglucose, 3-O-methylglucose and D-fucose exhibited a 10-30 fold preference for the high-affinity transporter.