Ime1 plays a pivotal role in the initiation of meiosis in a/alpha diploid cells of Saccharomyces cerevisiae. In the absence of glucose and nitrogen, IME1 expression is greater in a/alpha cells than in either a or alpha cells and therefore only a/alpha, but not a/a or alpha/alpha, cells are committed to sporulation. It is known that IME1 expression is positively regulated by Mck1, Rim1, Ime4 and the Swi-Snf complex but other factors may also be involved. In addition, Rme1 is assumed to repress IME1 expression. To provide more details of the repression of expression of IME1, we have isolated mutants in which the IME1p-PHO5 fusion gene integrated at the ura3 locus is expressed in alpha cells under nutritionally rich conditions. We found that mutations occurred in TUP1, SSN6, SIN4 and RGR1, among which TUP1 and SSN6 were identified for the first time as negative regulators of IME1 expression. Deletion of the Rme1-binding site from the IME1 promoter did not result in activation of the expression of IME1 under nutritionally rich conditions, suggesting that Rme1 does not function as a DNA-binding protein with the Tup1-Ssn6 repression complex. We also demonstrated that the 294-bp fragment from nucleotide position -914 to -621 and the 301-bp fragment from nucleotide position -1215 to -915 of the IME1 promoter region contain elements acting as URS and UAS in TUP1+ and tup1 mutant cells, respectively. These findings indicate that IME1 is negatively regulated by the Tup1-Ssn6 repressor complex through two distinct upstream regions in conjunction with unidentified DNA-binding proteins.