[en] The 5'-flanking region of the gene for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) is shown to contain promoter sequences that drive transcription in vitro in the presence of a HeLa whole-cell extract. DNase I protection studies revealed at least six different regions within the 277-base-pair (bp) promoter that bind nuclear proteins and produce footprints. The functional significance of these sequences was determined through transcriptional analysis of a series of substitution mutations that scrambled short sequences throughout this region. Two of the footprint sequences were crucial for transcription in vitro; one of these contains a match in 6 of 6 bp, with a sequence in the adenovirus type 2 major late promoter that is know to be required for transcription. Scrambling a 26-bp sequence in a third footprint led to a consistent 2-fold increase in transcription, suggesting that this sequence might be a site for negative regulation. These studies define three regions that play a role in regulating transcription of the gene for HMG-CoA reductase, a negatively regulated enzyme in the cholesterol biosynthetic pathway