Yarrowia lipolytica is a yeast which can utilize n-alkane as a sole carbon source. We isolated a Y. lipolytica peroxisomal acetoacetyl-CoA thiolase gene, PAT1, by complementation of a mutant that cannot utilize n-decane as a sole carbon source. We found that the putative PAT1 product had conserved features of peroxisomal acetoacetyl-CoA thiolase, We showed that the PAT1 disruptant was not able to grow on n-decane, and that n-decane-inducible acetoacetyl-CoA thiolase activity largely depended on PAT1, The original mutant carried a mutation involving the replacement of Gly382 with Glu. This mutation inactivated the ability of PAT1 to complement the defective n-decane utilization of the disruptant, These results indicate that PAT1 encodes peroxisomal acetoacetyl-CoA thiolase and is essential for n-decane utilization in Y. lipolytica,