Abstract Grg-1 is a Neurospora gene which was identified as being highly expressed glucose-repressible gene. A cis-acting regulatory region required for the regulated expression of grg-1 has been characterized. The regulatory region is found between 440 and 500 nucleotides upstream of the first major grg-1 start of transcription site and contains two distinct cis -acting regulatory elements. The upstream element consists of the sequence GTGACGTCAC, which is identical to the previously identified CRE (Cyclic AMP-Responsive Element). The second element is a newly defined cis -acting regulatory site. The element has the sequence TTGCTAGCAA and has been named NRS (Neurospora Repressor Site). DNA binding proteins can be shown to bind to both of these cis-acting regulatory elements. Experiments in which these sites were deleted demonstrate that both cis -acting regulatory elements are required to turn off the in vitro expression of the grg-1 gene under conditions of glucose sufficiency.