As part of a wider study on the molecular evolution of alanine:glyoxylate aminotransferase 1 (AGT1) intracellular compartmentalization, we have determined the subcellular distribution of immunoreactive AGT1, using postembedding protein A-gold immunoelectron microscopy, in the livers of various members of the classes Mammalia, Aves, and Amphibia. As far as organellar distribution is concerned, three categories could be distinguished. In members of the first category (type I), all, or nearly all, of the immunoreactive AGT1 was concentrated within the peroxisomes. In the second category (type II), AGT1 was found more evenly distributed in both peroxisomes and mitochondria. In the third category (type III), AGT1 was localized mainly within the mitochondria with much lower, but widely variable, amounts in the peroxisomes. Type I animals include the human, two great apes (gorilla, orangutan), two Old World monkeys (anubis baboon, Japanese macaque), a New World monkey (white-faced Saki monkey), a lago, morph (European rabbit), a bat (Seba's short-tailed fruit bat), two caviomorph rodents (guinea pig, orange-rumped agouti), and two Australian marsupials (koala, Bennett's wallaby). Type II animals include two New World monkeys (common marmoset, cotton-top tamarin), three prosimians (brown lemur, fat-tailed dwarf lemur, pygmy slow loris), five rodents (a hybrid crested porcupine, Colombian ground squirrel, laboratory rat, laboratory mouse, golden hamster), an American marsupial (grey short-tailed opossum), and a bird (raven). Type III animals include the large tree shrew, three insectivores (common Eurasian mole, European hedgehog, house shrew), four carnivores (domestic cat, ocelot, domestic dog, polecat ferret), and an amphibian (common frog). In addition to these categories, some animals (e.g. guinea pig, common frog) possessed significant amounts of cytosolic AGT1. Whereas the subcellular distribution of AGT1 in some orders (e.g. Insectivora and Carnivora) did not appear to vary markedly between the different members, in other orders (e.g. Primates, Rodentia and Marsupialia) it fluctuated widely between the different species. Phylogenetic analysis indicates that the subcellular distribution of AGT1 has changed radically on numerous occasions during the evolution of mammals. The new observations presented in this paper are compatible with our previous demonstration of a relationship between AGT1 subcellular distribution and either present or putative ancestral dietary habit, and our previous suggestion that the molecular evolution of the AGT gene has been markedly influenced by dietary selection pressure.