Kluyveromyces lactis, a budding yeast related to Saccharomyces cerevisiae, can grow on a wider variety of substrates and shows less sensitivity to glucose repression than does Saccharomyces cerevisiae. Many genes that are subject to glucose repression in S. cerevisiae are repressed only weakly or not at all in K. lactis. The molecular basis for this difference is largely unknown. To compare the mechanisms that regulate glucose repression in K. lactis and S. cerevisiae, we decided to clone and analyse an invertase gene from K. lactis. The SUC2 gene, which encodes invertase in S. cerevisiae, is strongly regulated by glucose and serves as a model system for studies on glucose repression. The invertase gene of K. lactis, KlINV1, was isolated by colony hybridization using a conserved region within the inulinase gene of K. marxianus as a probe. Two independent clones obtained were shown to contain the same ORF of 1827 bp. The deduced amino acid sequence is 59% similar to that of the K. marxianus inulinase and shows 49% similarity to ScSuc2p. Gene disruption experiments and low-stringency Southern analysis indicate that KlINV1 is a unique gene in K. lactis. Northern analysis revealed that the transcription of KlINV1 is strongly repressed in the presence of glucose, but, in contrast to the case in S. cerevisiae, repression is independent of KlMig1p.