The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD ؉ , with a intercept inhibition constant (K ii ) of 0.03 ؎ 0.004 M. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable -methylene derivatives of TAD or SAD were inhibitory, with K ii values 13-to 60-fold higher than that of mycophenolic acid.IMP dehydrogenase (IMPDH) converts IMP to XMP in the presence of NAD, a critical rate-limiting step in the biosynthesis of guanine nucleotides. Increased IMPDH activity is associated with actively dividing cells; consequently, IMPDH has been exploited as a target for anticancer, antiviral, immunosuppressive, and antimicrobial drug therapies (9, 15). Significant differences in IMDPH enzyme kinetics and inhibitor sensitivities between human and microbial homologues suggest that IMPDH may be an exploitable target for antimicrobials (25,36,40,42). Purine metabolic enzymes hold particular promise as targets to combat intracellular parasites while minimizing side effects to host cells. Parasites are inherently more reliant on these enzymes to maintain growth and are incapable of synthesizing purine precursors de novo (34).Toxoplasma gondii is an obligate intracellular protozoan responsible for fetal damage when the infection is acquired in utero (24), heart transplant complications (37), and opportunistic infections in immunocompromised individuals (39). T. gondii, as a member of the phylum Apicomplexa, is related to other parasites such as Plasmodium spp. (malaria) and Cryptosporidium spp. (26). A wide variety of molecular genetic tools have been developed for T. gondii (27,28). In particular, the generation of reagents for rever...