The existence of two distinct dihydrofolate reductases (DHFR) in highly trimethoprim-resistant clinical isolates has been unequivocally demonstrated. The enzymes have been characterized with regard to the affinity for substrates and sensitivity to inhibitors. The chromosomal, trimethoprim-sensitive DHFR was purified to homogeneity by a new simple two-step procedure. Its N-terminal amino acid sequence, determined up to the first 35 amino acids, showed 69% homology with the Escherichia coli DHFR.