Ammonia liberated during amino acid catabolism in mammals is highly neurotoxic and is detoxified by the five enzymes of the urea cycle that are expressed within the liver. Inborn errors of each of the urea cycle enzymes occur in humans. Carbamoyl phosphate synthetase I (CPSase I; EC 6.3.4.16) is located within the inner mitochondrial matrix and catalyzes the initial rate-limiting step of the urea cycle. Unless treated, complete deficiency of CPSase I, a rare autosomal recessive disease, causes death in newborn infants. Survivors are often mentally retarded and suffer frequent hyperammonemic crises during intercurrent illness or other catabolic stresses. Biochemically, CPSase I deficiency is characterized by high levels of blood ammonia, glutamine, and alanine, with low or absent citrulline and arginine levels. As a first step toward the development of gene therapy directed to the hepatocyte, we have generated a CPSase I-deficient mouse by gene targeting. Mice with homozygous disruption of CPSase I (CPSase [-/-] mice) die within 36 hours of birth with overwhelming hyperammonemia, and without significant liver pathology. This animal is a good model of human CPSase I deficiency.