We have determined the primary genomic and cDNA sequences encoding the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) enzyme of the protozoan parasite Toxoplasma gondii (dihydrofolate reductase, EC 1.5.1.3; thymidylate synthase EC 2.1.1.45). The DHFR-TS gene of T. gondii (strain RH) spans more than 6 kilobases of genomic DNA. Unlike the DHFR-TS genes of other protists, sequences encoding the Toxoplasma protein are interrupted by numerous intervening sequences. Analysis of processed T. gondii DHFR-TS cDNAs reveals a single open reading frame of 1830 nucleotides, predicting a 610-amino acid protein of molecular mass of 69 kilodaltons. Because its nucleotide composition and codon usage are roughly comparable to those observed in "higher" eukaryotes, the Toxoplasma DHFR-TS sequence is particularly useful for assessing evolutionary relationships between eukaryotic species. The predicted amino acid sequence for the DHFR-TS protein shows conservation of the major structural features identified in other DHFR and TS enzymes, while revealing certain differences which may be exploited for the design of novel antifolates for treatment of toxoplasmosis associated with AIDS.