We have determined the nucleic acid sequences of a gene encoding the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) from bovine and human AIDS isolates of Cryptosporidium parvum. THe DHFR-TS gene was isolated from genomic DNA libraries by hybridization with a probe amplified from C. parvum genomic DNA using generic TS primers in the polymerase chain reaction. Genomic Southern and electrophoretic karyotype analyses reveal C. parvum DHFR-TS is a single-copy gene on a 1200-kb chromosome. The DHFR-TS nucleic acid sequence contains no introns and the single 1563-bp open reading frame encodes a 179 residue N-terminal DHFR domain connected by a 55 amino acid junction peptide to a 287 residue C-terminal TS domain. The sequences of the DHFR-TS gene from the bovine and human C. parvum isolates differ at two positions in the 5'-flanking sequence and at 38 positions in the encoding sequence. These DNA sequence polymorphisms will provide a powerful probe to examine the genotypic diversity and genetic population structure of C. parvum. The two sequences encode identical TS domains which share all except one of the phylogenetically conserved amino acid residues identified among reported TS sequences. The predicted DHFR domain sequences contain nine amino acid differences; these polymorphisms all map to non-active site, surface locations in known DHFR structures. The C. parvum DHFR active site contains novel residues at several positions analogous to those at which point mutations have been shown to produce antifolate resistance in other DHFRs. Thus C. parvum DHFR may be intrinsically resistant ti inhibition by some antifolate DHFR inhibitors which may explain why cryptosporidiosis is refractory to treatment with the clinically common antibacterial and antiprotozoal antifolates.