We developed an assay which permits measurement of aspartate carbamoyltransferase (ACTase) activity. Cytosol from human peripheral blood mononuclear cells was used as the enzyme source. Using [14C]carbamoyl phosphate as the radiolabeled substrate, the formation of [14C]carbamoyl aspartate was quantitated by high performance liquid chromatography (HPLC) using an anion-exchange column with UV detection at 200-280 nm and an on-line liquid scintillation detector. A gradient method from an initially low concentration of ammonium phosphate, 1 mM (pH 3.0), to a higher concentration, 38 mM (pH 4.5), was used. The apparent Km values of carbamoyl phosphate and aspartate were 58 microM and 1.9 mM, respectively. ACTase inhibition by N-(phosphonacetyl)-l-aspartate (PALA) was consistent with a competitive model with respect to carbamoyl phosphate. The assay conditions were optimized to permit measurement of ACTase activity prior to and following therapy with PALA; ACTase was inhibited in a dose-dependent manner. This HPLC method permits direct quantitation of both the product of the reaction and the initial integrity of the substrate, [14C]carbamoyl phosphate, which is unstable in aqueous solutions.