In Crithidia fasciculata, carbamoyl phosphate synthetase II, which catalyses the first step of de novo pyrimidine biosynthesis, was separated from aspartate carbamoyltransferase by ammonium sulfate fractionation. The antitumor drug acivicin competitively inhibited the synthetase II activity with respect to L-glutamine, yielding an apparent Ki of 2 microM. In the absence of L-glutamine, acivicin resulted in a selective, time-dependent inactivation of L-glutamine-dependent activity of the enzyme, with an inactivation constant (Kinact) of 100 microM and a minimum inactivation half-time (T) of 0.2 min. L-Glutamine protected the enzyme from inactivation. These results are consistent with a postulate that acivicin is an active site-directed affinity analogue of L-glutamine, achieving irreversible inactivation. The inactivated enzyme retained ammonia-dependent activity. Acivicin stimulated the ammonia-dependent activity by increasing the Vmax value of the enzyme; apparent Km values for ammonia and MgATP were not affected. Differential action of acivicin on the Crithidia and mammalian synthetase II is discussed.