The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene from the monogenetic kinetoplastid protozoan, Crithidia fasciculata, was isolated and characterized. The gene is located on a single chromosome of approximately one megabase, and shows significant sequence similarity to other eukaryotic and prokaryotic DHFR and TS genes. There is a single low-abundance polyadenylated DHFR-TS transcript of approximately 3100 nt. One major miniexon splice site was identified by primer extension analysis. The 5' flanking region of the gene is divergently transcribed and shows strong similarities to a consensus DHFR promoter as well as to other eukaryotic 'housekeeping' gene promoter regions. A sequence downstream of the DHFR promoter consensus region is complementary to the 3' end of the C. fasciculata miniexon-derived RNA. This suggests a means by which the two separately transcribed RNAs may be juxtaposed for trans-splicing. In the 3' flanking region of the DHFR-TS gene, there is a sequence that is present in all of the chromosomes from this species and also from Leishmania tarentolae.