Although human alanine:glyoxylate aminotransferase (AGT) is imported into peroxisomes by a Pex5p-dependent pathway, the properties of its C-terminal tripeptide (KKL) are unlike those of any other type 1 peroxisomal targeting sequence (PTS1). We have previously suggested that AGT might possess ancillary targeting information that enables its unusual PTS1 to work. In this study, we have attempted to locate this information and to determine whether or not it is a characteristic of all vertebrate AGTs. Using the two-hybrid system, we show that human AGT interacts with human Pex5p in mammalian cells, but not yeast cells. Using (immuno)fluorescence microscopic analysis of the distribution of various constructs expressed in COS cells, we show the following. 1) The putative ancillary peroxisomal targeting information (PTS1A) in human AGT is located entirely within the smaller C-terminal structural domain of 110 amino acids, with the sequence between Val-324 and Ile-345 being the most likely candidate region. 2) The PTS1A is present in all mammalian AGTs studied (human, rat, guinea pig, rabbit, and cat), but not amphibian AGT (Xenopus). 3) The PTS1A is necessary for peroxisomal import of human, rabbit, and cat AGTs, but not rat and guinea pig AGTs. We speculate that the internal PTS1A of human AGT works in concert with the C-terminal PTS1 by interacting with Pex5p indirectly with the aid of a yet-to-be-identified mammal-specific adaptor molecule. This interaction might reshape the tetratricopeptide repeat domain allosterically, enabling it to accept KKL as a functional PTS1. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.