The general repression complex, constituted by the yeast Tup1 and Ssn6 factors, is a conserved global regulator of transcription in eukaryotes. In the yeast Saccharomyces cerevisiae, it is an important repressor of hypoxic genes, such as ANB1, under aerobic conditions and deletion of the TUP1 gene causes a flocculation phenotype. The KlTUP1 gene from the yeast Kluyveromyces lactis encodes for a protein with 83% similarity to Tup1 in S. cerevisiae. Despite the general domain conservation, the database searches showed the absence of a characteristic Tup1 glutamine-rich domain (Q1 at positions 96-116). Instead, there was a non-conserved sequence lacking the α-helix structure in this region. The ability to act as a transcriptional repressor was tested by expressing the KlTUP1 gene, in both high- and low-copy vectors, in an S. cerevisiae tup1 mutant strain. Repression effects were studied using the aerobic repressible reporter ANB1-lacZ and the effect on flocculation. In both regulatory systems, low levels of KlTup1 caused moderate (~30%) repression, but when the number of KlTup1 copies was increased, only the ANB1 reporter raised the repression levels of S. cerevisiae Tup1. These results show the capability of KlTup1 to act as a repressor in S. cerevisiae. The lower repression reached in S. cerevisiae is discussed in terms of structural differences.