The third step in the mitochondrial β-oxidation spiral of short-chain fatty acids is catalyzed by short-chain l-3-hydroxyacyl-CoA dehydrogenase (HADHSC; EC 1.1.1.35). We have determined the structural organization of the human HADHSC gene by sequencing of cloned genomic amplification products, obtained using HADHSC-specific cDNA-based primers, as well as by direct sequencing of an isolated PAC clone containing the HADHSC gene. Upon comparison with the HADHSC cDNA sequence, HADHSC was shown to encompass at least eight exons, ranging in size from 73 to 158 bp, and 7 introns. The total HADHSC gene spans approximately 49 kb. The HADHSC 5′-flanking region was characterized with an AluI plasmid library constructed from a partially AluI-digested PAC clone containing the human HADHSC gene. Several typical promoter elements such as a CAAT-box, Sp1, AP1, and AP2 sites were found, while a TATA-box was apparently absent. Among other putative regulatory elements, a NRRE-1 site was identified. By radiation hybrid panel, assisted fine-mapping HADHSC was linked to marker AFM070TH5, corresponding to Chromosome (Chr) 4q22-26, and a putative HADHSC pseudogene was linked to marker D15S1324, located at Chr 15q17-21. Knowledge of the genomic organization and 5′-flanking region of HADHSC will enable genomic mutation analysis of patients suspected of HADHSC deficiency, as well as facilitate the investigation into the transcriptional regulation of short-chain fatty acid oxidizing gene products in general and HADHSC expression in particular.