The ubiquitin encoding genes of Kluyveromyces lactis were cloned. Three genes, KlUBI1, KlUBI3 and KlUBI4, were found in this yeast, while in Saccharomyces cerevisiae there are four genes, UBI1, -2, -3 and -4. The UBI1/UBI2 duplication is thus absent from the K. lactis genome. General structural features of ubiquitin genes were very similar in these two species (presence of an intron in KlUBI1, fusion to ribosomal protein genes in KlUBI1 and KlUBI3, spacer-less polyubiquitin repeats in KlUBI4). Disruption or deletion of K. lactis ubiquitin genes showed that: (a) disruption of KlUBI1 was lethal (in S. cerevisiae, ubi1/ubi2 double deletion is lethal); (b) KlUBI3 is also an essential gene for cell growth; (c) deletion of KlUBI4 led to an increased sensitivity to high temperature, similar to the ubi4 mutation in S. cerevisiae, but, in contrast to the latter, the klubi4 mutant was not sensitive to carbon or nitrogen source starvation. The syntenic relationship of ubiquitin loci between K. lactis and S. cerevisiae genomes is also described.