The nucleotide sequence of a 3.4 kb fragment containing the HIS3 gene of the osmotolerant yeast Zygosaccharomyces rouxii has been determined. The fragment was cloned from a Z. rouxii genomic DNA library by complementation of a Saccharomyces cerevisiae his3 mutant strain. The sequenced DNA fragment contained three open reading frames; the middle one (678 bp long, predicting a protein of 226 amino acids) shared a high degree of similarity with HIS3 genes of other yeast species. In the promoter region of the putative ZrHIS3 gene, a T(c) element required for constitutive transcription was found. The GenBank Accession No. of the sequenced DNA region is Y18561.