The human gene encoding the bifunctional aminopeptidase and epoxide hydrolase enzyme, leukotriene A₄ hydrolase (LTA₄ hydrolase) has been cloned from a placental λ phage genomic library. The gene is greater than 35 kbp and contains 19 exons ranging in size over 24–312 bp. The introns range in size over 0.26–5.7 kbp. The essential zinc‐binding histidine residues and glutamate residue, which delineate the zinc‐binding domain required for both enzyme activities of LTA₄ hydrolase, are divided between exons 10 and 11. The LTA₄ hydrolase gene was localized to chromosome 12q22 utilizing fluorescence in situ hybridization. Based on the chromosome localization and genomic DNA analysis, LTA₄ hydrolase was determined to be a single‐copy gene. Primer‐extension analysis demonstrated that the transcription initiation site of LTA₄ hydrolase mRNA is 151 nucleotides upstream of the initiator ATG. Approximately 4 kbp of 5′‐flanking region of the LTA₄ hydrolase gene has been obtained and sequencing of 1.4 kb of this 5′‐flanking region demonstrated several transcription‐factor consensus sequences, including a phorbol‐ester‐response element (AP2) and two xenobiotic‐response elements. The cloning and characterization of the human gene for LTA₄ hydrolase provides a basis for further insight into transcriptional regulation of this bifunctional enzyme and its role in various inflammatory processes.