In most prokaryotic and eukaryotic organisms dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are encoded by independent genes. Evidence is presented here that the higher plant Arabidopsis thaliana has two bifunctional DHFR-TS genes. The structure of the genes, DHFR at the amino terminus and TS at the carboxy terminus, is identical to their organization in protozoa, the only other known organisms with bifunctional genes. Sequence alignments suggest that the bifunctional genes from protozoa and higher plants may have different evolutionary origins. The positions of the introns support the complementary hypothesis that the DHFR domain of the bifunctional plant genes and the monofunctional DHFR gene of vertebrates derive from a common, intron-containing progenitor, although the structure (bifunctional or monofunctional) of the ancestral gene remains indeterminate. Comparison of the two bifunctional genes of Arabidopsis indicates that the DHFR and TS domains evolved at different rates; each following the evolutionary history of their monofunctional counterparts. In contrast to the DHFR domain, the evolution of the TS domain shows a higher level of nucleotide and amino acid sequence conservation, but a remarkable variability in the intron positions.