The amino acid sequence of a trimethoprim-resistant dihydrofolate reductase (EC 1.5.1.3) specified by the R-plasmid R67 is described. The sequence was deduced from automatic and manual sequence analysis of the intact protein, the fragments produced by cyanogen bromide cleavage, and peptides derived from the largest cyanogen bromide fragment by digestion with trypsin, Staphylococcus aureus V8 proteus, chymotrypsin, and Lysobacter enzymogenes alpha-lytic protease. The complete sequence comprises 78 residues in a single polypeptide chain of molecular weight 8444. No evidence of heterogeneity was obtained, indicating that all subunits of the native enzyme are identical. Comparison of the sequence with that of all known dihydrofolate reductases shows no significant sequence homology.