The molecular basis of the variable species-specific peroxisomal and/or mitochondrial targeting of the enzyme alanine-glyoxylate aminotransferase 1 (AGT) has been studied in human fibroblasts by confocal immunofluorescence microscopy after intranuclear microinjection of various human, rabbit, marmoset, and feline AGT cDNA constructs. The expression of full-length human and rabbit AGT cDNA led to an exclusively peroxisomal distribution of AGT. However, the distribution of feline and marmoset AGT depended on the cDNA construct injected. In both species, injection of the short cDNAs (from transcripts that occur naturally in marmoset liver but not in feline liver) led to an exclusively peroxisomal distribution. However, injection of the long cDNAs (from transcripts that occur naturally in both species) led to most of the AGT being targeted to the mitochondria and only a small, yet significant, fraction to the peroxisomes. Reintroduction of the 'ancestral' first potential translation initiation site into human AGT cDNA led to an 'ancestral' distribution of AGT (i.e. both mitochondrial and peroxisomal). Deletion of the second potential translation start site from the long feline cDNA led to a distribution that was almost entirely mitochondrial, which suggests that most peroxisomal AGT encoded by the long cDNA results from internal translation initiation from this site with the consequent loss of the N-terminal mitochondrial targeting sequence. Expression of rabbit cDNA and the short marmoset and feline cDNAs in cells selectively deficient in the import of peroxisomal matrix proteins showed that peroxisomal AGT in all these species is imported via the peroxisomal targeting sequence type 1 (PTS1) import pathway. The almost complete functional dominance of the N-terminal mitochondrial targeting sequence over the C-terminal PTS. which was not due to any direct interference of the former with peroxisomal import, was maintained even when the unusual PTS1 of AGT (KKL in human) was replaced by the prototypical PTS1 SKL. The results demonstrate that the major determinant of alanine-glyoxylate aminotransferase subcellular distribution in mammals is the presence or absence of the mitochondrial targeting sequence rather than the peroxisomal targeting sequence. Various strategies have arisen during the evolution of mammals to enable the exclusion of the mitochondrial targeting sequence from the newly synthesised polypeptide, all of which involve the use of alternative transcription and/or translation initiation sites.