The Clorella kessleri HUP 1 gene coding for a hexose/H+ symporter has been expressed in a glucose uptake-deficient mutant of Schizosaccharomyces pombe. The transformants are able to grow on glucose and to accumulate 3-O-methylglucose 100-fold. This system has been used to test the activity of specifically mutated HUP 1 cDNAs. All three histidyl residues were exchanged with arginine (H73R, H170R, and H495R) without a major effect on transport activity. When Asp-44 within the first transmembrane helix was replaced by Asn, the transporter was inactive; replacement by Glu (D44E) resulted in a loss of activity by 90% and a 15-fold increased Km value. Glutamine residues conserved in all glucose transporters sequenced so far were exchanged: Q179N (in helix 5), Q298G and Q299N (both in helix 7). Whereas Q298G only resulted in a small Km change, both Q179N and Q299N showed an increase in Km by a factor of 10. Inserting 4 additional amino acids each into the two largest loops (1 and 6) reduced the activity dramatically; only in the latter case this was due to decreased protein synthesis or stability. Two COOH-terminal deletions (-27 and -43 amino acids) were also tested. The 27 COOH-terminal amino acids, but not the 43 COOH-terminal amino acids, could be removed without affecting transporter activity.