HMG-CoA reductase (HMGR) catalyzes the rate controlling step in cholesterol production. This enzyme is highly expressed in the liver where it is subject to extensive hormonal and dietary regulation. This study was undertaken to examine the occupancy and function of the hepatic HMGR promoter in regards to insulin and sterol regulation. HMGR protein and mRNA are substantially decreased in diabetic animals and rapidly restored by administration of insulin. Nuclear run-on assays revealed that HMGR transcription was substantially reduced in the diabetic rats, and fully restored within two hours after insulin treatment. In vivo footprinting revealed several areas of protein binding as shown by dimethyl sulfate protection or enhancement. The CRE was heavily protected in all conditions - including diabetes, cholesterol feeding, or statin treatment. Striking enhancements in footprints from diabetic animals were observed at -142 and at -161 (in the SRE). Protections at a newly ident ified NF-Y site at -70/-71 were seen in normal animals, and not in diabetics. This proximal NF-Y site was found to be required for efficient HMGR transcription. CREB-1 was able to bind the HMGR CRE in vitro, and to the promoter in vivo. The data supports an essential role for CREB in transcription of hepatic HMGR, and identifies at least two sites where in vivo occupancy is regulated by insulin. The technique of in vivo electroporation was utilized to perform the first functional analysis of the HMGR promoter in live animals. Analysis of a series of deletion constructs showed that deletion of the region containing the cyclic AMP response element (CRE) at -104 to -96 and the newly identified NF-Y site at -70 resulted in marked reduction of promoter activity. Possible sterol regulation of the promoter was investigated by raising tissue cholesterol levels by feeding cholesterol, or by inhibiting cholesterol synthesis with a statin (lovastatin). It was found that HMGR promoter constructs r esponded to lovastatin, in agreement with previous findings in cultured cells. This work sheds light on the regulation of the HMGR promoter in the liver, whose expression is a key determinant of serum cholesterol levels- a major risk factor for cardiovascular disease.