Diazepam, 7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2-one, is a widely used drug. It is used as an anticonvulsant, sedative, and muscle relaxant, and has a long duration of action (PubChem, 2011). The fungus Aspergillus niger J/8 N-demethylates diazepam to form 7-chloro-5-phenyl-3H-1,4-benzodiazepin-2-one. This demethylation step may be carried out by a transmethylase, hydrolytic demethylase or one of several different types of oxidative demethylases, so the generic "A" is used. The diazepin ring in the demethylated compound is then cleaved. It transiently forms 2-amino-N-(2-benzoyl-4-chlorophenyl)acetamide, which is N-acetylated to form N-(2-benzoyl-4-chlorophenyl)-2-acetamidoacetamide. The fungus Penicillium velutinum cleaves the diazepin ring at the same position without prior N-demethylation and performs the same N-acetylation, to form N-(2-benzoyl-4-chlorophenyl-2-acetamido-N-methylacetamide. Other fungi can demethylate diazepam, but this is the first report of fungal diazepam ring cleavage (Ambrus et al. 1975). A mixed microbial culture from activated sludge treatment has also been shown to demethylate diazepam to form 7-chloro-5-phenyl-3H-1,4-benzodiazepin-2-one (Helbling et al., 2010). The same culture also formed a mono-hydroxylated diazepam derivative, but the exact position of the hydroxyl group was not further specified.