Expression of the yeast genes DIT1 and DIT2 is confined to mid/late sporulation. Transcription of these two divergently arranged genes is controlled by a common 900-bp intergenic region. Random mutagenesis of this promoter and tests with appropriate reporter constructs identified an 11-bp cis-acting palindromic sequence, DIT repressor element (DRE), as a major negative regulatory site during vegetative growth. Repression is exerted by DRE in conjunction with a mid-sporulation element (MSE)-like sequence situated 26 bp away. These sequence elements are both contained within the 76-bp negative regulatory element (NRE) defined previously [Friesen H., Hepworth, S. R. & Segall, J. (1997) Mol. Cell. Biol. 17, 123-134]. The activated form of Rim101p, a transcriptional inducer of the early meiotic gene IME1, enhances expression from the DIT1 promoter both in vegetative and sporulating cells. Activation by Rim101p does not seem to involve binding of Rim101p at either of the two cis-acting sites described here, since reporter constructs with both elements or most of the NRE deleted could still be activated by the short form of Rim101p.