We have previously identified a series of triphenylmethane derivatives of deoxyuridine with antimalarial activity in vitro which selectively inhibit Plasmodium falciparum deoxyuridine triphosphate nucleotidohydrolase (PfdUTPase) compared to the human enzyme. The crystal structure of PfdUTPase in complex with one of these inhibitors suggested that the triphenylmethane derivative was selective due to a series of interactions between the trityl group and the side chains of residues Phe, Ile and Lys located in a hydrophobic pocket distinct from the phosphate binding site. Here we show by site-directed mutagenesis that the hydrophobic nature of the trityl binding site and in particular aromatic interactions established between the inhibitor and residue Phe contribute significantly to the binding of uracil-based derivatives containing trityl groups in the 5'-position. Thus, changing Phe for alanine resulted in increased K values for all compounds tested. Conversely, substitution of the polar residue Lys for Ala results in smaller K values and an increase in selectivity with regard to human dUTPase. This information will aid in the design of inhibitors with improved activity against the Plasmodium enzyme.