A conserved tripeptide serves as a peroxisomal targeting signal (PTS) for proteins. The firefly luciferase protein contains a PTS at its COOH terminus, consisting of the tripeptide serine-lysine-leucine. Site-directed mutagenesis showed that this tripeptide is essential for peroxisomal import. Mutations in this tripeptide abolished its function, indicating that the PTS is a conserved sequence. The study also demonstrated that the tripeptide serine-lysine-leucine is sufficient to direct a cytosolic protein into peroxisomes. Additional experiments revealed that the tripeptide is a common feature of many peroxisomal proteins, suggesting that it is a conserved targeting signal. In contrast, other protein transport processes, such as those into mitochondria, chloroplasts, or the ER, use larger, non-conserved signals that are cleaved upon import. The unique properties of peroxisomal protein import suggest a different translocation mechanism. The study also identified additional PTSs in other peroxisomal proteins, including human catalase, pig D-amino acid oxidase, rat acyl-CoA oxidase, and rat enoyl-CoA/hydratase-3-hydroxyacyl-CoA dehydrogenase. The PTS is located at the COOH terminus of these proteins, and its presence is necessary for their import into peroxisomes. The study provides evidence that the PTS is a conserved sequence and is essential for peroxisomal protein import. The results suggest that the PTS is a fundamental element in peroxisomal protein targeting and that its conservation across species indicates a universal mechanism for peroxisomal import.A conserved tripeptide serves as a peroxisomal targeting signal (PTS) for proteins. The firefly luciferase protein contains a PTS at its COOH terminus, consisting of the tripeptide serine-lysine-leucine. Site-directed mutagenesis showed that this tripeptide is essential for peroxisomal import. Mutations in this tripeptide abolished its function, indicating that the PTS is a conserved sequence. The study also demonstrated that the tripeptide serine-lysine-leucine is sufficient to direct a cytosolic protein into peroxisomes. Additional experiments revealed that the tripeptide is a common feature of many peroxisomal proteins, suggesting that it is a conserved targeting signal. In contrast, other protein transport processes, such as those into mitochondria, chloroplasts, or the ER, use larger, non-conserved signals that are cleaved upon import. The unique properties of peroxisomal protein import suggest a different translocation mechanism. The study also identified additional PTSs in other peroxisomal proteins, including human catalase, pig D-amino acid oxidase, rat acyl-CoA oxidase, and rat enoyl-CoA/hydratase-3-hydroxyacyl-CoA dehydrogenase. The PTS is located at the COOH terminus of these proteins, and its presence is necessary for their import into peroxisomes. The study provides evidence that the PTS is a conserved sequence and is essential for peroxisomal protein import. The results suggest that the PTS is a fundamental element in peroxisomal protein targeting and that its conservation across species indicates a universal mechanism for peroxisomal import.