The gene encoding the liver-specific peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT, EC. 18.104.22.168) exists as two common polymorphic variants termed the "major" and "minor" alleles. The P11L amino acid replacement encoded by the minor allele creates a hidden N-terminal mitochondrial targeting sequence, the unmasking of which occurs in the hereditary calcium oxalate kidney stone disease primary hyperoxaluria type 1 (PH1). This unmasking is due to the additional presence of a common disease-specific G170R mutation, which is encoded by about one third of PH1 alleles. The P11L and G170R replacements interact synergistically to reroute AGT to the mitochondria where it cannot fulfill its metabolic role (i.e. glyoxylate detoxification) effectively. In the present study, we have reinvestigated the consequences of the interaction between P11L and G170R in stably transformedCHOcells and have studied for the first time whether a similar synergism exists between P11L and three other mutations that segregate with the minor allele (i.e. I244T, F152I, and G41R). Our investigations show that the latter three mutants are all able to unmask the cryptic P11L-generated mitochondrial targeting sequence and, as a result, all are mistargeted to the mitochondria. However, whereas the G170R, I244T, and F152I mutants are able to form dimers and are catalytically active, theG41Rmutant aggregates and is inactive. These studies open up the possibility that all PH1 mutations, which segregate with the minor allele, might also lead to the peroxisome-to- mitochondrion mistargeting of AGT, a suggestion that has important implications for the development of treatment strategies for PH1. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.