Using a Yarrowia lipolytica genomic library, several overlapping clones of the peroxisomal 3-oxoacyl-CoA-thiolase gene, POT1, were isolated. The library was prepared in the bacterial expression vector lambda gt11, thus allowing an immunological screening of recombinant bacteriophages with specific antibodies raised against purified peroxisomal thiolase. The isolated POT1 clones hybridized to a 1.4 kb RNA species, which was induced approximately 30-fold when oleate was the carbon source. A 3634-bp segment of the cloned DNA was sequenced. This segment contained, on both strands, three major overlapping open-reading frames of 678, 1122 and 1242 bp. Northern-hybridization analysis showed that only the largest of these reading frames was transcribed. It encodes a protein of 414 amino acids and molecular mass 43.059 kDa. Its deduced amino acid sequence has 30-60% identity and 50-70% sequence similarity when compared to other known thiolases. According to both the amount (68-71%) and location of conserved amino acids, the encoded protein belongs to the peroxisomal rather than the mitochondrial or cytoplasmic class of thiolases. Compared to bacterial and yeast cytosolic thiolases, the POT1 gene product contains a N-terminal extension of 25 amino acids which clearly differs from typical mitochondrial import signals. One of the isolated clones contained, in addition to the POT1 coding sequence, 784 bp of the corresponding 5' flanking region. Nevertheless, it was efficiently expressed in Escherichia coli suggesting the correct recognition of this fungal promoter by the prokaryotic transcriptional and translational machinery. The Y. lipolytica genomic POT1 gene was disrupted by replacing 120 bp of its coding sequence with 2.7 kbp of DNA including the Y. lipolytica LEU2 gene. The resulting delta pot1::LEU2 cells were free of immunologically cross-reacting thiolase. Western-blot analysis showed that the product of the non-disrupted gene had a molecular mass of approximately 42 kDa. This corresponds well to the molecular mass of purified Y. lipolytica peroxisomal thiolase. Disruption of POT1 abolished the ability of Y. lipolytica cells to grow on solid media with oleate as a carbon source. This inability to grow in the presence of oleate suggests both the catabolic function of POT1 and the absence of additional catabolic thiolases in Y. lipolytica. However, the delta pot1::LEU2 cells were unaffected in their ability to elongate externally added tridecanoic acid to its higher-chain-length homologues. Hence, another, POT1-independent and biosynthetic 3-oxoacyl-CoA thiolase must be responsible for this reaction in Y. lipolytica.