Two different NAD/coenzyme-dependent formaldehyde dehydrogenases exist, the well-known NAD/GSH-dependent (EC 1.2.1.1) and the more recently discovered NAD/Factor-dependent enzyme. The GSH-dependent one has been found in eukaryotes and Gram-negative bacteria, the Factor-dependent one in two different Gram-positive bacteria. Previous work also showed that Factor and GSH are not interchangeable in the enzymatic reactions. Here it is revealed that the Factor is identical to mycothiol (MySH), 1-O-(2'-[N-acetyl-L-cysteinyl]-amido-2'-deoxy-alpha-D-glucopyranosyl)-D- myo-inositol, a thiol compound which has recently been detected in Actinomycetes. Thus, MySH is GSH's companion as it is the coenzyme for the enzyme which henceforth can be indicated as NAD/MySH-dependent formaldehyde dehydrogenase.