Abstract β-TGDDF, the displacement product of 2-thioacetamide ribonucleotide and N-10-(bromoacetyl)-5,8-dideazafolate, is a potent, slow, tight-binding, multisubstrate adduct inhibitor (MAI) of glycinamide ribonucleotide transformylase (GAR TFase: E.C. 18.104.22.168.). The mechanism of inhibition by this MAI and its derivatives are reported. In addition, a related series of MAIs formed from the interaction of glycinamide ribonucleotide (GAR) or its carbocyclic analog (carbo-β-GAR) and N-10-(bromoacetyl)-5,8-dideazafol with GAR TFase have been discovered and characterized. These latter enzyme assembled inhibitors represent a novel route to the inhibition of GAR TFase.