The primary structure of dihydrofolate reductase from bovine liver has been established by Edman degradation of the intact carboxymethylated protein and of peptides obtained from the protein by the action of cyanogen bromide, trypsin, and the protease from Staphylococcus aureus, respectively. Since separation of some of the peptide mixtures by classical methods proved impossible, new systems were developed for the use of high-performance liquid chromatography to separate such mixtures. Some of the cleavage procedures used to obtain peptides gave atypical results at certain peptide bonds. The results are discussed in terms of the residues involved in these unexpectedly resistant or sensitive bonds. The sequence of the bovine liver enzyme is compared with those published for the enzyme from other sources, and known or probable functions of invariant residues are described. Sequences of vertebrate and bacterial reductases are compared and contrasted, and a possible role is considered for the residues which are invariant in bacterial reductases, but different in vertebrate reductases, in determining the selective inhibitory action of trimethoprim on bacterial reductases.