A key enzyme in the mitochondrial beta-oxidation of unsaturated fatty acids is the 3,2-trans-enoyl-CoA isomerase (DCI; EC 188.8.131.52). It catalyzes the transformation of 3-cis and 3-trans intermediates arising during the stepwise degradation of all cis-, mono-, and polyunsaturated fatty acids to the 2-trans-enoyl-CoA intermediates. A genomic clone encoding the human DCI was isolated and characterized by use of the previously cloned human DCI cDNA. The entire gene encompasses approximately 12.5 kb, and the coding sequence is distributed over seven exons. One major and three minor transcription start sites were determined by primer extension analysis. In common with promoters of other housekeeping genes encoding mitochondrial proteins, the GC-rich, immediate 5'-flanking region of the DCI transcription initiation site lacks typical TATA and CAAT boxes; instead, two GC box consensus sequences are present. Introns 2 and 6 contain several Alu repetetive sequences. The human DCI gene locus was assigned to chromosome 16 by use of human-rodent somatic cell hybrids and to chromosome 16p13.3 by chromosomal in situ suppression hybridization studies.