The cluster of three genes, ACR1, ACR2, and ACR3, previously was shown to confer arsenical resistance in Saccharomyces cerevisiae. The overexpression of ACR3 induced high level arsenite resistance. The presence of ACR3 together with ACR2 on a multicopy plasmid was conducive to increased arsenate resistance. The function of ACR3 gene has now been investigated. Amino acid sequence analysis of Acr3p showed that this hypothetical protein has hydrophobic character with 10 putative transmembrane spans and is probably located in yeast plasma membrane. We constructed the acr3 null mutation. The resulting disruptants were 5-fold more sensitive to arsenate and arsenite than wild-type cells. The acr3 disruptants showed wild-type sensitivity to antimony, tellurite, cadmium, and phenylarsine oxide. The mechanism of arsenical resistance was assayed by transport experiments using radioactive arsenite. We did not observe any significant differences in the accumulation of 76AsO33- in wild-type cells, acr1 and acr3 disruptants. However, the high dosage of ACR3 gene resulted in loss of arsenite uptake. These results suggest that arsenite resistance in yeast is mediated by an arsenite transporter (Acr3p).