Sporulation of the yeast Saccharomyces cerevisiae is a process of cellular differentiation that occurs in MATa/MAT alpha diploid cells in response to starvation. The sporulation-specific genes DIT1 and DIT2, which are required for spore wall formation, are activated midway through the sporulation program, with maximal transcript accumulation occurring at the time of prospore enclosure. In this study, we have identified a negative regulatory element, termed NREDIT, that is located between the start sites of transcription of these divergently transcribed genes. This element, which prevents expression of the DIT1 and DIT2 genes during vegetative growth, reduces expression of a CYC1-lacZ reporter gene more than 1,000-fold and acts in an orientation- and position-independent manner. We found that the ability of NREDIT to turn of expression of the reporter gene and the chromosomal DIT1 and DIT2 genes in vegetative cells requires the Ssn6-Tup1 repression complex. Interestingly, NREDIT-mediated repression of the reporter gene is maintained during sporulation. Derepression during sporulation requires complex interactions among several cis-acting elements. These are present on an approximately 350-bp DNA fragment extending from NREDIT to the TATA box and an approximately 125-bp fragment spanning the TATA box of DIT1. Additionally, a region of NREDIT which is very similar in sequence to UASSPS4, an element that activates gene expression midway through sporulation, contributes both to vegetative repression and to sporulation-specific induction of DIT1. We propose a model to explain the requirement for multiple elements in overcoming NREDIT-mediated repression during sporulation.