The Kluyveromyces lactis FUS1 gene was cloned, physically characterized and its role in the mating response pathway was determined. The gene encodes a putative membrane protein, whose structure shows a single membrane-spanning segment, a short extracellular amino-terminus and a long carboxy-terminus, located in the cytoplasmic side. The predicted primary structure of the protein shows a number of serine and threonine residues in the amino-terminus, which in analogy to Fus1p of Saccharomyces cerevisiae might be O-glycosylated. A fus1-GFP hybrid protein was tentatively located in the plasma membrane of dividing cells and upon activation of the pheromone response pathway, the protein seems to be relocated at the tip of elongated cells. KlFus1p is required for optimal conjugation of sexual partners and its expression is significantly enhanced by overexpression of both a constitutively active form of KlGpa1p, the G protein alpha subunit that triggers the mating response in this strain, and the KlSte12p transcription factor. Inactivation of the KlSte12 protein strongly reduces mating and affects KlFUS1 gene expression. The KlFUS1 gene has been deposited in the GenBank under accession number AF519444.