Intracellular cadmium (Cd(2+)) ion accumulation and the ability to produce specific Cd(2+) ion chelators was studied in the methylotrophic yeast Hansenula polymorpha. Only one type of Cd(2+) intracellular chelators, glutathione (GSH), was identified, which suggests that sequestration of this heavy metal in H. polymorpha occurs similarly to that found in Saccharomyces cerevisiae, but different to Schizosaccharomys pombe and Candida glabrata which both synthesize phytochelatins. Cd(2+) ion uptake in the H. polymorpha wild-type strains appeared to be an energy dependent process. It was found that Deltagsh2 mutants, impaired in the first step of GSH biosynthesis, are characterized by increase in net Cd(2+) ion uptake by the cells, whereas Deltagsh1/Deltamet1 and Deltaggt1 mutants impaired in sulfate assimilation and GSH catabolism, respectively, lost the ability to accumulate Cd(2+) intracellularly. Apparently H. polymorpha, similarly to S. cerevisiae, forms a Cd-GSH complex in the cytoplasm, which in turn regulates Cd(2+) uptake. Genes GSH1/MET1 and GGT1 are involved in maturation and metabolism of cellular Cd-GSH complex, respectively. Transport of [(3)H]N-ethylmaleimide-S-glutathione ([(3)H]NEM-SG) conjugate into crude membrane vesicules, purified from the wild-type cells of H. polymorpha appeared to be MgATP dependent, uncoupler insensitive and vanadate sensitive. We suggest that MgATP dependent transporter involved in Cd-GSH uptake in H. polymorpha, is similar to S. cerevisiae Ycf1-mediated vacuolar transporter responsible for accumulation of organic GS-conjugates and Cd-GSH complex.