The gene encoding arachidonate 12-lipoxygenase was cloned from a porcine EMBL3 genomic library using a cDNA probe of the enzyme, and its nucleotide sequence was determined. The gene consists of 14 exons with 13 introns, and spans approximately 8 kilobases. Analysis of splice junctions indicated that all of the splice donor and acceptor sites conformed to the GT/AG rule. An approximately 1-kilobase region upstream of the coding sequence contains nine GC-boxes for potential Sp1-binding sites at positions -77, -135, -145, -165, -214, -636, -643, -684, and -813. There are two sets of AP-2 binding sequence at positions -234 and -402. Neither typical TATA box nor CCAAT box is found in this region. The transcriptional start site was determined by primer extension analysis, and was tentatively identified as a cytidine residue located 19 bases upstream from initiation codon. Southern blot analysis revealed the presence of one copy of 12-lipoxygenase gene per haploid genome. We found striking similarities in genomic organization as well as the promoter sequences between the porcine 12-lipoxygenase and the rabbit 15-lipoxygenase genes, suggesting that these genes are evolutionarily related.