We have determined the crystal structure of dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Cryptosporidium hominis, revealing a unique linker domain containing an 11-residue ␣-helix that has extensive interactions with the opposite DHFR-TS monomer of the homodimeric enzyme. Analysis of the structure of DHFR-TS from C. hominis and of previously solved structures of DHFR-TS from Plasmodium falciparum and Leishmania major reveals that the linker domain primarily controls the relative orientation of the DHFR and TS domains. Using the tertiary structure of the linker domains, we have been able to place a number of protozoa in two distinct and dissimilar structural families corresponding to two evolutionary families and provide the first structural evidence validating the use of DHFR-TS as a tool of phylogenetic classification. Furthermore, the structure of C. hominis DHFR-TS calls into question surface electrostatic channeling as the universal means of dihydrofolate transport between TS and DHFR in the bifunctional enzyme.Thymidylate synthase (TS) 1 and dihydrofolate reductase (DHFR) are essential enzymes in the cell cycle of all organisms, since they catalyze the production of dTMP, required for DNA replication. TS converts the substrate, dUMP, to dTMP by reductive methylation using the cofactor, 5,10-methylene tetrahydrofolate, and releases dihydrofolate (1). In the presence of the cofactor NADPH, DHFR reduces dihydrofolate to tetrahydrofolate. The folate cycle is completed by serine hydroxymethyl transferase, which converts tetrahydrofolate back to 5,10-methylene tetrahydrofolate.Recently, Stechmann and Cavalier-Smith (2) have addressed the problem of locating the root of the eukaryotic tree, one of the most challenging evolutionary problems. In several protozoa, including Alveolates and Euglenozoa, and in some plants, the genes for DHFR and TS are translated as a single polypeptide, forming a bifunctional enzyme (DHFR-TS), whereas in most animals, fungi, and bacteria, these two enzymes are monofunctional. The monofunctional form of DHFR is a monomer, and that of TS is a dimer. The currently held hypothesis is that the primordial form of DHFR and TS is the monofunctional form and that the genes for DHFR and TS became fused at a single evolutionary point. If the DHFR-TS gene fusion occurred just once, then the fused gene provides an excellent phylogenetic marker, since reversing the fusion would require multiple genetic events. Stechmann and Cavalier-Smith have used the derived gene fusion between DHFR and TS to place the root of the tree below the common ancestor of plants, Alveolates, and Euglenozoa (Fig.