The complete nucleotide and encoded amino acid sequences were determined for the dihydrofolate reductase (DHFR) from the bacteria Enterobacter aerogenes and Citrobacter freundii. These were compared with the closely related Escherichia coli DHFR sequence. The ancestral DHFR sequence common to these three species was reconstructed. Since that ancestor there have been seven, nine, and one amino acid replacements in E. coli, E. aerogenes, and C. freundii, respectively. In E. coli, five of its seven replacements were located in the beta-sheet portion of the protein, and all seven were located in a single restricted region of the protein. In E. aerogenes, all nine of its replacements were located within surface residues, with five clustered in a region topologically distinct from the E. coli cluster. The replaced side chains are sometimes in direct contact but more often are separated by an intervening side chain. It is argued that the temporal clustering of replacements is typical for the evolution of most proteins and that the associated topological clustering gives a picture of how evolutionary change is accommodated by protein structure.