The Kluyveromyces lactis genes for sexual pheromones have been analyzed. The a-factor gene encodes a predicted polypeptide of 187 amino acid residues containing four tridecapeptide repeats (WSWITLRPGQPIF). A nucleotide BLAST search of the entire K. lactis genome sequence allowed the identification of the nonannotated putative a-pheromone gene that encodes a predicted protein of 33 residues containing one copy of the dodecapeptide a-factor (WIIPGFVWVPQC). The role of the K. lactis structural genes KIMF alpha 1 and KIMFA1 in mating has been investigated by the construction of disruption mutations that totally eliminate gene functions. Mutants of both alleles showed sex-dependent sterility, indicating that these are single-copy genes and essential for mating. MAT alpha Klsst2 mutants, which, by analogy to Saccharomyces cerevisiae, are defective in G alpha-GTPase activity, showed increased sensitivity to synthetic a-factor and increased capacity to mate. Additionally, Klbar1 mutants (putatively defective in a-pheromone proteolysis) showed delay in mating but sensitivity to a-pheromone. From these results, it can be deduced that the K. lactis MATa cell produces the homolog of the S. cerevisiae a-pheromone, whereas the MATa cell produces the a-pheromone.