Abstract A procedure has been developed for the purification of lipoamide dehydrogenase, glutathione reductase, thioredoxin reductase, and thioredoxin from Escherichia coli B, in which the four proteins are purified together in the initial stages separation effected in two steps, and the individual purifications completed in one or two steps. The extinction coefficient of FAD in thioredoxin reductase has been determined. Amino acid analyses of lipoamide dehydrogenase and thioredoxin reductase are given. The half-cystine content of the E. coli lipoamide dehydrogenase is only one-half of that found with the pig heart enzyme.