Chromosomes XII and XIII of a Saccharomyces carlsbergensis brewing strain were analysed after their transfer into Saccharomyces cerevisiae by kar1-mediated single chromosome transfer. The lager yeast was found to be heterozygous for the isoleucine-valine biosynthesis genes IL V2 (encoding acetohydroxy acid synthase) and IL V5 (encoding acetohydroxy acid reductoisomerase). In both cases, Southern analysis showed restriction site polymorphisms, and that one allele hybridizes more strongly to that of S. cerevisiae than the other. The alleles with limited nucleotide sequence homology are located on chromosomes which recombine poorly with the corresponding S. cerevisiae chromosomes (XIII and XII) during meiosis. A cluster of ribosomal RNA genes is located on the chromosome XII with the S. cerevisiae-like IL V5, but not on the homoeologous chromosome. The present analysis supports the view that S. carlsbergensis is an amphiploid hybrid.