Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolates on its enzyme activity, as well as on in vitro parasite growth. The full-length gene consists of a 1,548-bp open reading frame encoding a 58.8-kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. Each domain contained active-site amino acid residues responsible for the enzymatic activity. The expressed soluble recombinant DHFR-TS protein was approximately 57 kDa after glutathione S-transferase (GST) cleavage, similar to an approximately 58-kDa native enzyme identified from the parasite merozoite. The non-GST fusion recombinant DHFR enzyme revealed K(m) values of 4.70 +/- 0.059 (mean +/- standard error of the mean) and 9.75 +/- 1.64 microM for dihydrofolic acid (DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC(50)] = 68.6 +/- 5.20 nM) than pyrimethamine (IC(50) = 55.0 +/- 2.08 microM) and trimethoprim (IC(50) = 50 +/- 12.5 microM). Moreover, the antifolates' inhibitory effects on DHFR enzyme activity paralleled their inhibition of the parasite growth in vitro, indicating that the B. gibsoni DHFR could be a model for studying antifolate compounds as potential drug candidates. Therefore, the B. gibsoni DHFR-TS is a molecular antifolate drug target.