Mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) synthase, a liver-specific enzyme, is a constituent of the HMG-CoA cycle responsible for ketone-body synthesis. We report the isolation and characterization of genomic clones that encompass the gene for rat mitochondrial HMG-CoA synthase. The gene spans at least 24 kbp and contains ten exons and nine introns. The 5' flanking region of the gene has also been cloned and characterized. Exon 1 contains the untranslated sequence of the transcript, extending downstream to enclose the coding region for the putative mitochondrial-targeting signal (35 amino acids). The 1149-bp proximal region of the transcription start point permits transcription of a reporter gene in transfected hepatoma cells but not in an extrahepatic cell line, confirming the function of the promoter. A truncated construct of 142 bp is still able to promote transcription in hepatoma cells, suggesting the presence of liver-specific enhancer elements in the proximal promoter region. The 5' flanking region contains typical promoter elements, including a TATA box and several putative recognition sequences for transcription factors involved in controlling both basal-level and hormone-modulated transcription rates. Furthermore, the presence in the mitochondrial HMG-CoA-synthase promoter of cis-elements, responsible for the multihormonal regulation of transcription, is supported by transient transfection experiments.