To investigate the mechanism of induction of the peroxisomal beta-oxidation enzymes by peroxisome proliferators, genes of these enzymes were cloned from rat liver and their structures analyzed. The acyl-CoA oxidase gene was found to produce two forms of the enzyme differing in their amino acid sequences in a limited region, through alternative splicing of the two copies of the third exon. The amino acid sequence of the bifunctional enzyme suggests, compared with those of its mitochondrial counterparts, that the enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities are located on the amino- and the carboxyl sides, respectively. Two copies of the 3-ketoacyl-CoA thiolase gene were identified per haploid genome. One gene named A is constitutive and encodes a thiolase precursor carrying 36 amino acid residues of amino-terminal presequence, whereas the other, termed B, is remarkably induced by peroxisome proliferators and specifies a precursor having a 26-residue presequence. Functional analysis of the upstream sequence of the acyl-CoA oxidase gene revealed three functionally different regions, one of which had the character of ciprofibrate-responsive enhancer. In this region, two sequences were identified as binding-sites of rat liver nuclear proteins. A gene transfection study indicated that these sequence elements (termed A and B) play important roles in the induction of the gene, the former acting positively whereas the latter probably is acting negatively.