The complete gene for human fatty acid ethyl ester Synthase-III (FAEES-III) was isolated from a human genomic lambda phage library for functional and structural determination. The gene spans approximately 3.3 kb which includes 791 base pairs of the 5' and 124 base pairs of the 3' flanking regions. The gene is comprised of seven exons and is interrupted by six introns. Several transcription regulatory sequences were identified in the promoter region. Primer extension experiments demonstrated the existence of two possible transcription initiation sites at nucleotide -29 and 32 position, 5' to the start of the translation. In addition to a TATA box at position -29 relative to the transcription initiation site and two Spl GGGCGG recognition sequences at nucleotide positions -42 to -37 and -50 to -45, the promoter contains a sequence motif matching the transcription activating factor AP-1. We also found an A + T rich region between nucleotide -505 and -390 which contained twenty-two AAAAT tandem repeats. The gene for FAEES-III was localized to human chromosome 11 by hybridizing the genomic fragment Xh01 to Chinese hamster/human somatic cell hybrid panels. These data extend our knowledge of non-oxidative alcohol metabolism and permit linkage analyses between this pathway and alcohol-related phenotypes.