Ornithine carbamoyltransferase has been purified from the liver of the loggerhead turtle Caretta caretta by a single-step procedure using chromatography on an affinity column to which the transition-state analogue, delta-N-(phosphonoacetyl)-L-ornithine (delta-PALO), was covalently bound. The procedure employed yielded an enzyme which was purified 373-fold and was judged to be homogeneous by nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed a specific activity of 224. The molar mass of the C. caretta enzyme was approximately 112 kDa, the single band obtained by SDS-PAGE indicated a subunit molar mass of 39.5 kDa; hence, the enzyme is a trimer of identical subunits. It catalyzes an ordered sequential mechanism in which carbamoyl phosphate binds first, followed by L-ornithine. The Michaelis constants were 0.858 mM for L-ornithine and 0.22 mM for carbamoyl phosphate, the dissociation constant of the enzyme-carbamoyl phosphate complex was 0.50 mM.