We have previously characterized, in Trypanosoma brucei, a multigene family encoding two developmentally regulated glucose transporters that are 80% identical at the amino-acid level. We report here the characterization of the homologous glucose transporters (TcoHT1 and TcoHT2) in Trypanosoma congolense, an African trypanosome responsible for disease in domestic animals. Both TcoHT isoforms, which are 92.4% identical, are encoded by a single cluster of genes containing two copies of TcoHT1 and three copies of TcoHT2 arranged alternately. Northern blot analysis revealed that TcoHT2 is expressed in all of the adaptive forms, while mRNA encoding TcoHT1 is only present in the metacyclic and bloodstream forms of T. congolense. When transfected with the TcoHT2 gene, Chinese Hamster Ovary cells express a hexose transporter with properties similar to those of the T. congolense procyclic forms (Km D-glucose = 41 microM versus 64 microM). In contrast to TcoHT2, TcoHT1 expressed in the Chinese hamster ovary cells appeared to be a relatively low affinity glucose transporter (Ki D-glucose = 0.8 mM). To determine the region(s) involved in the different apparent affinities for glucose, a chimera analysis was undertaken on the TcoHT isoforms. This study shows that amino-acid residues important for D-glucose recognition are located in the central region (between transmembrane domains 3 and 7) and in the C-terminal intracellular domain of TcoHT2. Site directed mutagenesis identified Ser193 located within transmembrane helix 4 as a key residue in relaxing the apparent affinity of TcoHT1 for glucose.