The unicellular green alga Chlorella kessleri can induce monosaccharide-H+ symport catalyzing the energy-dependent transport of D-glucose (D-Glc) and several other pentoses and hexoses across the plasmalemma. The gene coding for the inducible HUP1 monosaccharide-H+ symporter has been cloned and the protein has been characterized previously. The data presented in this paper demonstrate that the presence of the HUP1 gene product alone is not sufficient to cover the broad substrate specificity of monosaccharide transport in induced Chlorella cells. Two other HUP genes are shown to be co-induced in Chlorella in response to D-Glc in the medium. The cloning of HUP2 and HUP3 cDNA and genomic sequences is described, both being very homologous to HUP1. Modification of the 5' untranslated sequences of full-length cDNA clones of HUP2 and HUP3 allowed the functional expression of both transporters in Schizosaccharomyces pombe. HUP2 was shown to be a galactose-H+ symporter, whereas the substrate specificity of the HUP3 gene product is very similar to that of the HUP1 protein. However, HUP3 does not seem to be induced to high levels in Glc-treated Chlorella cells. Results are also presented proving that the product of the HUP1 gene is localized in the plasmalemma of D-Glc-induced Chlorella cells and is absent in plasma membranes of noninduced cells. Incubation of thin sections of Chlorella cells with anti-HUP1 antibodies and a fluorescence-labeled, second antibody yielded a ring of fluorescence on the surface of Glc-induced Chlorella cells.