Acetoacetyl-CoA thiolase is an enzyme that catalyses both the CoA-dependent thiolytic cleavage of acetoacetyl-CoA and the reverse condensation reaction. In Dictyostelium discoideum, acetoacetyl-CoA thiolase (DdAcat) is encoded by a single acat gene. The aim of this study was to assess the localization of DdAcat and to determine the mechanism of its cellular localization. Subcellular localization of DdAcat was investigated using a fusion protein with GFP, and it was found to be localized to peroxisomes. The findings showed that the targeting signal of DdAcat to peroxisomes is a unique nonapeptide sequence (15RMYTTAKNL23) similar to the conserved peroxisomal targeting signal-2 (PTS-2). Cell fractionation experiments revealed that DdAcat also exists in the cytosol. Distribution to the cytosol was caused by translational initiation from the second Met codon at position 16. The first 18 N-terminal residues also exhibited function as a mitochondrial targeting signal (MTS). These results indicate that DdAcat is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol using both PTS-2 and MTS signals, which overlap each other near the N-terminus, and the alternative utilization of start codons.