Overlapping genomic clones containing the entire sequence of the rat acyl-CoA oxidase gene were isolated and characterized. This gene spans about 25 kilobases, and there are 14 exons and 13 introns. All of the exon-intron junction sequences agree with the GT/AG rule. The results of Southern blot analyses indicated that the gene occurs once per haploid genome. In a preceding paper (Miyazawa, S., Hayashi, H., Hijikata, M., Ishii, N., Furuta, S., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8131-8137), we described the presence of two species of acyl-CoA oxidase mRNA which have different sequences, only in a small region. We identified two exons corresponding to this region. The above two mRNA species are produced by alternative splicing of these exons. Several mRNA cap sites were detected by S1 mapping and by the primer extension method. The principal one was mapped at 75 nucleotides upstream of the initiator methionine codon. The 5′-flanking region of the acyl-CoA oxidase gene was sequenced up to about 1300 nucleotides, upstream of the cap sites. There was neither a “TATA” box nor a typical “CCAAT” box sequence, at least up to nucleotide −500, rather, this region is G + C-rich (65%). “GC” box hexanucleotides (GGGCGG or CCGCCC) repeat six times in this region. Two of them, located nearest the cap sites, are in a complete 11-base pair direct repeat, 5′-GGGCGGGGCCG-3′. The features of the 5′-flanking sequence of this gene resemble those of the gene for rat enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. The possible functional significance of the characteristic 5′-flanking sequence elements in the transcriptional regulation of acyl-CoA oxidase is given attention.