Aspergillus sp. P37 is an arsenate-resistant fungus isolated from a river with a long history of heavy metal contamination. Its hypertolerant mechanism relies on an efficient arsenate reduction system in conjunction with increased coupling capabilities to glutathione and translocation of these complexes into the vacuolar system. Here, the comparison of arsenic resistance in two Aspergillus species and Saccharomyces cerevisiae in the presence or absence of different osmolytes revealed a link between tolerance to osmotic stress and tolerance to arsenic in Aspergillus strains. Under osmotic stress generated by NaCl or KCl, both Aspergillus strains were less resistant to arsenite and arsenate. In the presence of sorbitol, Aspergillus strains were more resistant to arsenite. Intracellular accumulation of arsenic was affected by the presence of the osmolytes (NaCl or sorbitol), suggesting a major role for vacuoles in the detoxification of arsenic in Aspergillus.