ABSTRACT.The pathway of de novo pyrimidine biosynthesis in the rodent parasitic protozoaBabesia rodhainihas been investigated. Specific activities of five of the six enzymes of the pathway were determined: aspartate transcarbamylase (ATCase: E.C. 2.1.3.2): dihydroorotase (DHOase: E.C. 3.5.2.3): dihydroorotate dehydrogenase (DHO‐DHase: E.C. 1.3.3.1); orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10); and orotidine‐5′‐phosphate decarboxylase (ODCase: E.C. 4.1.1.23). Michaelis constants for ATCase, DHO‐DHasc. OPRTase, and ODCase were determined in whole homogenates. Several substrate analogs were also investigated as inhibitors and inhibitor constants determined. N‐(phosphonacetyl)‐L‐aspartate was shown to be an inhibitor of the ATCase with an apparent K, of 7μM. Dihydro‐5‐azaorotate inhibited the DHO‐DHase (K, 16 μM) and 5‐azaorotate (Ki, 21 μM) was an inhibitor of the OPRTase. The UMP analog, 6‐aza‐UMP (Ki, 0.3 μM) was a potent inhibitor of ODCase, while lower levels of inhibition were found with the product. UMP (Ki, 120 μM) and the purine nucleotide, XMP (K1, 95 μM). Additionally, menoctone, a ubiquinone analo