Unlike most mitochondrial matrix proteins, the mitochondrial 3-oxoacyl-CoA thiolase [EC 2.3.1.16] is synthesized with no cleavable presequence and possesses information for mitochondrial targeting and import in the mature protein This mitochondrial thiolase is homologous with the mature portion of peroxisomal 3-oxoacyl-CoA thiolase and acetoace-tyl-CoA thiolase [EC 2.3.1.9] of Zoogloea ramigera along the entire sequence A hybrid gene encoding the NH₂-terminal 16 residues (MALLRGVFIVAAKRTP) of the mitochondrial thiolase fused to the mature portion of rat ornithine carbamoyltransferase [EC 2.1.3.3] (lacking its own presequence) was transfected into COS cells, and subcellular localization of the fusion protein was analyzed Cell fractionation and immunocytochemical analyses showed that the fusion protein was localized in the mitochondria These results indicate that the NH₂-terminal 16 residues of the mitochondrial thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein The fusion protein containing the NH₂-terminal 14 residues (MSTPSIVIASARTA) of the bacterial thiolase was also localized in the mitochondria On the other hand, the fusion protein containing the corresponding portion (MQASASDVVVVHGQRTP.) of the peroxisomal thiolase appeared not to be localized to the mitochondria These results show that the import signal of mitochondrial 3-oxoacyl-CoA thiolase originated from the NH₂-terminal portion of the ancestral thiolase The ancestral enzyme might have already possessed a mitochondrial import activity when mitochondria appeared first, or that it might have acquired the import activity during evolution by accumulation of point mutations in the NHL-terminal portion of the enzyme.