Two genes, MFal and MFa2, coding for the a-factor in yeast Saccharomyces cerevisiae were identified by in situ colony hybridization to synthetic probes to a yeast genomic library. The probes were designed on the basis of the known amino acid sequence of the tridecapeptide a-pheromone. The nucleotide sequence revealed that the two genes, though similar in their overall structure, differ from each other in several striking ways. MFal gene contains 4 copies of the coding sequence for the a-factor, which are separated by 24 nucleotides encoding the octapeptide Lys-Arg-Glu-Ala-Glu(or Asp)-Ala-Glu-Ala. The first a-factor coding block is preceded by a sequence for the hexapeptide Lys-Arg-Glu-Ala-Glu-Ala and 83 additional amino acids. MFa2 gene contains coding sequences for two copies of the a-factor that differ from each other and from a-factor encoded by MFal gene by a Gln → Asn and a Lys → Arg substitution. The first copy of the a-factor is preceded by a sequence coding for 87 amino acids which ends with Lys-Arg-Glu-Ala-Val-Ala-Asp-Ala. The coding blocks of the two copies of the pheromone are separated by the sequence for Lys-Arg-Glu-Ala-Asn-Ala-Asp-Ala. Thus, the a-factor can be derived from 2 different precursor proteins of 165 and 120 amino acids containing, respectively, 4 and 2 copies of the pheromone.