A flocculation gene was cloned from a Saccharomyces cerevisiae ATCC60715 genomic library, known to contain the FLO8 gene, on the basis of its ability to confer a flocculation phenotype on a nonflocculent strain. From a total of 11 130 clones, four clones sharing the several restriction fragments were isolated, suggesting that these were derived from the same locus. The results of integration mapping and disruption of the cloned gene indicated that this gene was the FLO8 gene. After disruption of the FLO8 gene, the strain lost its ability to flocculate. The DNA sequence of the FLO8 gene was determined. This gene includes a 2187-bp open reading frame that encodes a 729-amino acid protein. Computer analysis indicated that the FLO8 gene has a significant degree of homology with a S. cerevisiae chromosome V DNA sequence, but no homology with the FLO1 gene. The hydrophobicity profile of the putative FLO8 gene product did not indicate the presence of any significantly hydrophobic regions. Southern analysis of the FLO8 gene present in various yeast strains indicated that the FLO8 gene is highly conserved in yeast strains having a variety of flocculation phenotypes and genotypes. Northern analysis revealed that the level of FLO1 gene transcription is dependent on the rate of transcription of the FLO8 gene. These results suggest that the FLO8 gene mediates flocculation via transcriptional activation of the FLO1 gene.