A structurally diverse library of 93 lipophilic di- and tricyclic diaminopyrimidine derivatives was tested for the ability to inhibit recombinant dihydrofolate reductase (DHFR) cloned from human and bovine isolates of Cryptosporidium parvum (J. R. Vásquez et al., Mol. Biochem. Parasitol. 79:153-165, 1996). In parallel, the library was also tested against human DHFR and, for comparison, the enzyme from Escherichia coli. Fifty percent inhibitory concentrations (IC₅₀s) were determined by means of a standard spectrophotometric assay of DHFR activity with dihydrofolate and NADPH as the cosubstrates. Of the compounds tested, 25 had IC₅₀s in the 1 to 10 µM range against one or both C. parvum enzymes and thus were not substantially different from trimethoprim (IC₅₀s, ca. 4 µM). Another 25 compounds had IC₅₀s of <1.0 µM, and 9 of these had IC₅₀s of <0.1 µM and thus were at least 40 times more potent than trimethoprim. The remaining 42 compounds were weak inhibitors (IC₅₀s, >10 µM) and thus were not considered to be of interest as drugs useful against this organism. A good correlation was generally obtained between the results of the spectrophotometric enzyme inhibition assays and those obtained recently in a yeast complementation assay (V. H. Brophy et al., Antimicrob. Agents Chemother. 44:1019-1028, 2000; H. Lau et al., Antimicrob. Agents Chemother. 45:187-195, 2001). Although many of the compounds in the library were more potent than trimethoprim, none had the degree of selectivity of trimethoprim for C. parvum versus human DHFR. Collectively, the results of these assays comprise the largest available database of lipophilic antifolates as potential anticryptosporidial agents. The compounds in the library were also tested as inhibitors of the proliferation of intracellular C. parvum oocysts in canine kidney epithelial cells cultured in folate-free medium containing thymidine (10 µM) and hypoxanthine (100 µM). After 72 h of drug exposure, the number of parasites inside the cells was quantitated by indirect immunofluorescence microscopy. Sixteen compounds had IC₅₀s of <3 µM, and five of these had IC₅₀s of <0.3 µM and thus were comparable in potency to trimetrexate. The finding that submicromolar concentrations of several of the compounds in the library could inhibit in vitro growth of C. parvum in host cells in the presence of thymidine (dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors, in combination with leucovorin, may find use in the treatment of intractable C. parvum infections.