The NAD(P)H:flavin oxidoreductase (NADPH:riboflavin oxidoreductase) from Escherichia coli, Fre, is a monomer of 26.1 kDa, which catalyzes the reduction of free flavins by NADPH or NADH. A sequential ordered mechanism, with NADPH binding first, operates. Fre is the prototype of a class of flavin reductases able to transfer electrons with no prosthetic group. It has been previously reported that several members of this family, including Fre, were inactivated by thiol reagents such as N-ethylmaleimide (MalNEt). Amino acid sequence similarities among these enzymes reveal that one of the three cysteines residues of Fre is highly conserved. Altogether this suggested a crucial role of cysteine residues for catalysis. The three cysteine residues were mutated to serine residues. Single-mutant and double-mutant enzymes were as active as the wild-type and Km values for both substrates remained the same. Cysteine residues are thus not important for activity. Nevertheless, we showed that cysteines 5 and 214, but not cysteine 149, were responsible for MalNEt inactivation. In addition, it has been found that riboflavin, but not NADPH, can protect Fre from MalNEt inactivation. This strongly suggested that Cys5 and Cys214 are located at the flavin-binding site of Fre and that flavin can bind to the enzyme in the absence of NADPH.