The Saccharomyces cerevisiae PNT1 gene was isolated and characterized. When present in high copy number in S. cerevisiae, PNT1 confers resistance to the anti-Pneumocystis carinii drug pentamidine. The PNT1 gene encodes a previously uncharacterized polypeptide of 409 amino acids. The predicted gene product is a very basic (pI 9.9) polypeptide with one potential membrane-associated region. PNT1 is located on chromosome XVR of S. cerevisiae. It is transcribed at a very low level. Overexpression of the gene increases resistance to the cytostatic and mitochondrial DNA-damaging effects of pentamidine and related cationic compounds. Disruption of the gene leads to slightly increased levels of susceptibility to pentamidine and some related compounds.