The cDNAs HUP1 and HUP2 of Chlorella kessleri code for monosaccharide/H+ symporters that can be functionally expressed in Schizosaccharomyces pombe. By random mutagenesis three HUP1 mutants with an increased Km value for D-glucose were isolated. The 40-fold increase in Km of the first mutant is due to the amino acid exchange N436I in putative transmembrane helix XI. Two substitutions were found in a second (G97C/I303N) and third mutant (G120D/F292L), which show a 270-fold and 50-fold increase in Km for D-glucose, respectively. An investigation of the individual mutations revealed that the substitutions I303N and F292L (both in helix VII) cause the Km shifts seen in the corresponding double mutants. These mutations together with those previously found support the hypothesis that helices V, VII, and XI participate in the transmembrane sugar pathway. Whereas for most mutants obtained so far the Km change for D-glucose is paralleled by a corresponding change for other hexoses tested, the exchange D44E exclusively alters the Km for D-glucose. Moreover the pH profile of this mutant is shifted by more than 2 pH units to alkaline values, indicating that the activity of the transporter may require deprotonation of the corresponding carboxyl group. Chimeric transporters were constructed to study the 100-fold lower affinity for D-galactose of the HUP1 symporter as compared with that of the HUP2 protein. A crucial determinant for the differential D-galactose recognition was shown to be associated with the first external loop. The effect could be pinpointed to a single amino acid change: replacement of Asn-45 of HUP1 with isoleucine, the corresponding amino acid of HUP2, yields a transporter with a 20 times higher affinity for D-galactose. The reverse substitution (I47N) decreases the affinity of HUP2 for D-galactose 20-fold.