Here we use gene targeting to disrupt multiple enzymes in the parasite’s purine and pyrimidine nucleotide pathways, two of which, dihydrofolate reductase-thymidylate synthase (DHFR-TS) (11) and inosine monophosphate dehydrogenase (IMPDH) (12), are currently pursued as targets for the treatment of cryptosporidiosis. Here, TYMS is linked to cryptosporidiosis.