Mitochondrial glutamine synthetase (EC 6.3.1.2) is the primary ammonia-detoxifying enzyme in avian liver and is therefore analogous in function to carbamoyl-phosphate synthetase I (ammonia) (EC 6.3.4.16) in mammalian liver. In mammalian liver, glutamine synthetase is cytosolic and its distribution is restricted to a few hepatocytes around the terminal venules. These cells do not express carbamoyl-phosphate synthetase I. Using immunocytochemistry, we show here that there is little or no zonation of glutamine synthetase in avian liver. Rather, it is broadly distributed to most hepatocytes, much like carbamoyl-phosphate synthetase I in mammalian liver. In situ hybridization with a cloned glutamine synthetase cDNA probe showed the distribution of glutamine synthetase mRNA in both mammalian and avian liver to correspond to the distribution of immunoreactive protein. Neither glutamine synthetase nor carbamoyl-phosphate synthetase I and ornithine transcarbamoylase (EC 2.1.3.3) are strictly zoned in liver of the Texas tortoise or of an Argentine tree frog, both of which possess a complete urea cycle but which may also rely on glutamine synthetase for ammonia detoxication. These latter results suggest that the mutually exclusive expression of either carbamoyl-phosphate synthetase I or glutamine synthetase may be unique to mammalian liver.