Ammonia- and N-acetylglutamate-dependent carbamyl phosphate synthetase-I (EC 2.7.2.5), the mitchondrial enzyme involved in the initial step of urea biosynthesis, was purified to homogeneity from frog liver and crystallized. The purification involved extraction of a particulate fraction with cetyltrimethylammonium bromide in the presence of the protease inhibitors antipain, leupeptin, chymostatin, and pepstatin; acetone precipitation; and affinity chromatography with Cibacron blue F3GA-coupled agarose. The enzyme was adsorbed to the gel at pH 8.3 in the presence of 5 mM MgCl2 and eluted with magnesoum-free buffer. The enzyme crystallized as either elongated, thin, rectangular plates or as clusters of small crystals from 37 to 40% saturated ammonium sulfate. The enzyme moved as a single polypeptide band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis with a molecular weight of 160,000. In the absence of protease inhibitors, proteolysis of the enzyme occurred with the formation of an enzymatically active fragment with a subunit molecular weight of 139,000.