The product of the porcine HSD17B4 gene is a peroxisomal 80 kDa polypeptide containing three functionally distinct domains. The N-terminal part reveals activities of 17beta-estradiol dehydrogenase type IV and D-specific 3-hydroxyacyl CoA dehydrogenase, the central part shows D-specific hydratase activity with straight and 2-methyl-branched 2-enoyl-CoAs. The C-terminal part is similar to sterol carrier protein 2. The 80 kDa polypeptide chain ends with the tripeptide AKI, which resembles the motif SKL, the first identified peroxisome targeting signal PTS1. So far AKI, although being similar to the consensus sequence PTS1, has neither been reported to be present in mammalian peroxisomal proteins, nor has it been shown to be functional. We investigated whether the HSD17B4 gene product is targeted to peroxisomes by this C-terminal motif. Recombinant human PTS1 binding protein Pex5p interacted with the bacterially expressed C-terminal domain of the HSD17B4 gene product. Binding was competitively blocked by a SKL-containing peptide. Recombinant deletion mutants of the C-terminal domain lacking 3, 6, and 14 amino acids and presenting KDY, MIL, and IML, respectively, at their C-termini did not interact with Pex5p. The wild-type protein and mutants were also transiently expressed in the HEK 293 cells. Immunofluorescence analysis with polyclonal antibodies against the C-terminal domain showed a typical punctate peroxisomal staining pattern upon wild-type transfection, whereas all mutant proteins localized in the cytoplasm. Therefore, AKI is a functional PTS1 signal in mammals and the peroxisome targeting of the HSD17B4 gene product is mediated by Pex5p.