In Saccharomyces spp. the ability to use melibiose depends on the presence of a MEL gene encoding alpha-galactosidase. We used two cloned MEL genes as probes to characterize the physical structure and chromosomal location of the MEL genes in several industrial and natural Mel+ strains of Saccharomyces cerevisiae, Saccharomyces pastorianus, and Saccharomyces bayanus. Electrokaryotyping showed that all of the S. pastorianus strains and most of the S. bayanus strains studied had one MEL locus. The MEL gene in S. bayanus strains was similar but not identical to the S. pastorianus MEL gene. Mel+ S. cerevisiae strains had one to seven loci containing MEL sequences. The MEL genes of these strains could be divided into two categories on the basis of hybridization to MEL1, one group exhibiting strong hybridization to MEL1 and the other group exhibiting weak hybridization to MEL1. In S. pastorianus and S. bayanus strains, the MEL gene was expressed as a single 1.5-kb transcript, and the expression was galactose inducible. In some S. cerevisiae strains, the MEL genes were expressed even without induction at fairly high levels. Expression was usually further induced by galactose. In two strains, CBS 5378 and CBS 4903, expression of the MEL genes was at the same level without induction as it was in most other strains with induction. In all S. cerevisiae strains, irrespective of the number of MEL genes, mRNA of only one size (1.6 kb) was observed.