Publisher Summary S-Formylglutathione is the product of the oxidation of formaldehydeby the glutathione- and NAD-dependent formaldehyde dehydrogenase. S-Formylglutathione does not accumulate in crude tissue preparations, but is rapidly hydrolyzed. A specific enzyme, S-formylglutathione hydrolase, is responsible for most of the hydrolysis of this thiol ester. S-Formylglutathione hydrolase can be assayed by recording the absorbance decrease resulting from the hydrolysis of the thiol ester with a spectrophotometer at 240 nm and 25°. The assay can also be used for crude preparations. From each of the sources that are mentioned in the chapter, the high activity of the enzyme allows the use of highly diluted preparations, which diminishes interference from the high absorbance of crude preparations. Purification procedure initially involves the extraction of the tissue, salt precipitation, and diethylaminoethyl (DEAE)-cellulose chromatography. Further process includes washing on hydroxylapatite column, isoelectric focusing, and washing on Sephadex G-100 column. The final preparation is homogeneous by electrophoretic and ultracentrifugal criteria and represents about 2300-fold purification over the crude soluble fraction of the human liver.