Gliotoxin is an epipolythiodioxopiperazine produced by the opportunistic fungal pathogen Aspergillus fumigatus. It contains an intact disulphide bridge, which mediates its toxic effects via redox cycling. Gliotoxin biosynthesis is directed by the gli gene cluster, and knowledge of the biosynthetic pathway which leads to gliotoxin formation is limited, although LPhe and L-Ser are known amino acid precursors and gliT is a gliotoxin oxidoreductase responsible for self-protection and disulphide bridge closure. Deletion of gliG, herein shown to be an epoxide-conjugating glutathione stransferase, from the gli cluster results in loss of gliG expression and the complete abrogation of gliotoxin biosynthesis. Instead, this deletion mutant, A. fumigatus !gliG, secretes a 6-benzyl-6-hydroxy-1-methoxy-3- methylenepiperazine-2,5-dione (262.1026 u), herein identified and structurally characterized for the first time, which is proposed to be a shunt metabolite formed in the absence of gliG. This putative shunt metabolite contains a hydroxyl group at C-6, consistent with a gliotoxin biosynthetic pathway involving thiolation, which is mediated by the addition of the glutathione thiol group to a reactive acyl imine intermediate. A new reduction and alkylation assay, which uses sodium borohydride and 5’-iodoacetamidofluorescein to label gliotoxin, yields a stable, labelled gliotoxin product, di-acetamidofluoresceingliotoxin (GT-(AF)2; 1103.47 Da). This species is readily detectable by RPHPLC and exhibits a 6.8-fold increase in molar absorptivity compared to gliotoxin, which results in a higher sensitivity of detection (50 ng; 125 pmol). Unlike gliotoxin, GT-(AF)2 is detectable by MALDI-ToF MS. 6-benzyl-6- hydroxy-1-methoxy-3-methylenepiperazine-2,5-dione cannot be alkylated and so is devoid of thiols or a disulphide bridge. Complementation of gliG restored gliG expression and gliotoxin production which coincided with the disappearance of 6-benzyl-6-hydroxy-1-methoxy-3-methylenepiperazine-2,5- dione. In addition, gliG was confirmed, unlike gliT, not to be involved in selfprotection against gliotoxin. It is over 75 years since gliotoxin was discovered. The work presented herein provides the first evidential support of the thiolation mechanism leading to gliotoxin biosynthesis, in addition to confirming a novel biosynthetic role for a glutathione s-transferase in fungi.