The rate-limiting enzyme of cholesterol biosynthesis, HMG CoA reductase, is controlled by negative feedback regulation of transcription. We have isolated the reductase gene from a bacteriophage lambda genomic library prepared from hamster UT-1 cells. The 25 kilobase gene is split into 20 exons. The 5' untranslated and promoter regions differ from those of previously characterized genes. The 5' untranslated region encompasses as many as 670 nucleotides; contains up to eight AUG codons upstream of the codon used to initiate translation; and has multiple transcription initiation sites as determined by S1 nuclease mapping and primer extension analysis. The promoter region lacks a characteristic TATA box and CCAAT box; is rich in G + C residues (65%); and contains repeat sequences homologous to the 21 base pair repeats of the SV40 promoter. These unusual features may be relevant to the mechanism of expression of "housekeeping" genes, particularly those that are subject to negative feedback regulation.