Dihydrofolate reductase from an amethopterin-resistant strain of Streptococcus faecium contains a single cysteine residue per mole of protein (mol. wt. 20,000). Treatment of the enzyme with either DTNB or pHMB results in inactivation. The loss of enzyme activity correlates with the modification of 1.0 cysteine residue. Either of the substrates, TPNH or dihydrofolate, afford complete protection against inactivation by DTNB. Decreases in protein fluorescence emission and in the aromatic side-chain Cotton effect exhibited by the chemically altered enzyme suggest that the cysteine residue may occupy a part of the hydrophobic region near one or more tryptophan residues previously implicated in substrate binding.