Summary. Kinetic parameters on dihydroorotate dehydrogenase (DHO-DHase) from the rodent malarial parasite, Plasmodium berghei, have been determined. This enzyme, the fourth in de novo pyrimidine biosynthesis, is particulate and is absent in the mature mammalian red cell. The K,,, of the substrate, dihydroorotate, was determined to be 23 MM and the Ki values for a number of substrate analogues have been determined. The most potent inhibitor was dihydroazaorotate (Kl, 3 MM). The product orotate was also a good inhibitor (Ki, 5 MM) as were methylorotate (Ki, 10 /"M), 5-azaorotate (Ki, 20 MM) and other pyrimidine analogues. The activity of the enzyme was also affected by a number of respiratory chain inhibitors.As the P. berghei infection is accompanied by reticulocytosis, a comparative study of DHO-DHase in mouse reticulocytes was also carried out. The general properties of the enzyme from these sources were similar to those of the parasite enzyme. However, significant differences in the response of the two enzymes to various inhibitors were observed and could provide a rational basis for the development of chemotherapeutic agents active against the parasite.