Peroxisomal acyl-CoA oxidase (ACOX; EC 1.3.3.6) is the first enzyme of the fatty acid beta-oxidation pathway, which catalyzes the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, and it donates electrons directly to molecular oxygen, thereby producing H2O2. The discovery of carcinogenic peroxisome proliferators, which markedly increase the levels of this H2O2-producing ACOX in rat and mouse liver, generated interest in peroxisomal beta-oxidation system genes. The present study deals with the structural organization of human ACOX gene. This gene spans approximately 33 kb and consists of 14 exons and 13 introns. Primer-extension analysis revealed three principal cap sites, which were mapped at 50, 52, and 53 nt upstream of the initiator methionine codon. The 5' flanking region of the ACOX gene was sequenced up to 500 bp upstream of the cap sites. This promoter region is G + C-rich and contains three copies of the "GC box" hexanucleotides. Multiple GC boxes are a characteristic feature of the rat ACOX and bifunctional protein genes of the beta-oxidation system. A + T-rich TATA-boxlike sequences, TTTATTT and TTATT, have also been identified in this human ACOX gene, but typical CCAAT motifs are absent. This ACOX gene has been mapped to chromosome 17q25 by in situ hybridization, using a biotinlabeled probe.