Nucleotide sequence analysis of a cDNA clone shown to direct the synthesis in Escherichia coli of a pI 6.5 form of dihydrofolate reductase (DHFR) with an apparent molecular weight of 21,000 has clarified the allelic nature of the DHFR genes present in the Chinese hamster lung cell line DC-3F. By comparison with other cDNAs encoding different forms of DHFR produced by these cells or by antifolate-resistant sublines derived from them (Melera, P.W., Davide, J.P., Hession, C.A., and Scotto, K.W (1984) Mol. Cell. Biol. 4, 38-48) and with the use of transcription vectors to generate homogeneous populations of specific DHFR mRNAs for subsequent translation in vitro, we demonstrate that, with respect to the proteins they encode, these alleles differ only at amino acid position 95; a conversion of Asp----Asn at this position is solely responsible for the electrophoretic mobility and pI differences between the Mr 21,000 pI 6.5 and Mr 20,000 pI 6.7 forms of the enzyme. We also show that the conversion of Leu to Phe at position 22 of the Mr 21,000 pI 6.5 enzyme results in a mutant form whose catalytic activity is equal to or greater than normal, but whose IC50 for methotrexate is 85 microM. Additionally, the in vitro translation experiments show that the minor pI forms of DHFR known to exist in Chinese hamster lung cells are generated by a translational or post-translational modification step. Preliminary evidence suggests that this modification may result from an acetylation of the N terminus of the protein.