The HUP1 gene codes for the monosaccharide/H+ cotransporter protein of Chlorella kessleri. The gene is functionally expressed in Schizosaccharomyces pombe. This heterologous system has been used to screen for Km mutants of the Chlorella symporter. Since S. pombe transformed with HUP1 cDNA showed a 1000-fold increase in sensitivity toward the toxic sugar analogue 2-deoxyglucose, we screened for transformants with a decreased 2-deoxyglucose sensitivity. The transformants were produced with HUP1 cDNA randomly mutagenized by PCR. From 73 transformants with decreased 2-deoxyglucose sensitivity, four mutants with increased Km values for D-glucose were obtained. The amino acid exchanges responsible for the increased Km values are located in the center of the putative transmembrane helices V (Q179E), VII (Q298R), and XI (V433L/N436Y). Q179N and Q299N had previously been shown by directed mutagenesis to affect the Km value of the transporter for D-glucose. The drastic mutational changes Q298R and N436Y gave rise to very high Km values; however, the corresponding conservative amino acid changes Q298N or N436Q obtained by directed mutagenesis also result in Km values increased by a factor of 10 or 20, respectively. The data therefore support the proposal that at least helices V, VII, and XI may line the sugar translocation path and determine its specificity. These results are discussed in relation to other sugar transporters and to the interaction of the yeast hexokinase B with D-glucose as known from published crystal structures.