Northern analysis showed that DNA from the flocculation gene FLO1 hybridized to mRNA molecules of 4.8 kb. This transcript was specific for the FLO1 gene at the right end of chromosome I since disruption of this gene resulted in the disappearance of the transcript. We further found an absolute correlation between flocculation and the presence of transcripts hybridizing to FLO1 DNA, both in various flocculent and non-flocculent strains and in cells from the non-flocculating and flocculating stages of growth. In all cases transcripts were present in flocculating and absent from non-flocculating cultures. From these results we conclude that the FLO1 gene is transcriptionally regulated. Mutations in TUP1 or SSN6 cause flocculation. Several transcripts hybridizing to FLO1 DNA were present in the mutants but not in the corresponding wild-type strains. Disruption of the FLO1 gene in the tup1 and ssn6 strains showed that one of the transcripts corresponded to the FLO1 gene. Disruption of FLO1 did not abolish flocculation completely but only reduced it, indicating that at least two flocculation genes, including FLO1, are activated or derepressed by mutations in the TUP1/SSN6 regulatory cascade.