This paper describes a structure-activity study to identify novel, small-molecule inhibitors of the enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) from parasitic protozoa. The successful synthesis of a variety of analogues of dUMP is described in which the substituents are introduced at the 3'- and 5'-positions, together with variation in the heteroatom at the 5'-position. The compounds were assayed against recombinant Plasmodium falciparum and Leishmania major enzymes and the human enzyme to give a measure of selectivity. The compounds were also tested in vitro against the intact parasites P. falciparum and L. donovani. A number of potent and selective inhibitors of the P. falciparum dUTPase that show drug-like properties and represent good leads for future development were identified. The best inhibitors included the compounds 5'-tritylamino-2',5'-dideoxyuridine (2j) (Ki = 0.2 microM) and 5'-O-triphenylsilyl-2',3'-didehydro-2',3'-dideoxyuridine (5h) (Ki = 1.3 microM), with selectivity greater than 200-fold compared to the human enzyme. Structural features important for antiplasmodial activity were determined. The correlation observed between the inhibition of the enzyme and the inhibition of the parasite growth in vitro demonstrates that the P. falciparum dUTPase constitutes a valid and attractive novel target for the development of much-needed new antimalarial drugs.