Isoniazid(INH),the most-widely used anti-tuberculosis drug,has been shown to be activated by catalase-peroxidase enzyme(KatG)and Mn(Ⅱ)to produce the reactive carbon-centered isonicotinic acyl radical,which was considered to be responsible for its anti-tuberculosis activity.However,it is still not clear whether the previously-proposed N-centered isoniazidyl radical intermediate can be initially produced or not; and if so,what is its exact location on the hydrazine group,distal-or proximalnitrogen? Through complementary applications of ESR spin-trapping and HPLC/MS methods,here we show that the characteristic and transient N-centered isoniazidyl radical intermediate can be detected and identified from INH activation by the unique Mn(Ⅱ)Acetate.The exact location of the radical was found to be at the distal-nitrogen of the hydrazine group by 15N-isotope-labeling techniques via using 15N-labeled INH.Diisonicotinyl hydrazine was identified as a new reaction product from INH/Mn(Ⅱ).Analogous results were observed with other hydrazides.This study represents the first detection and unequivocal identification of the initial N-centered isoniazidyl radical and its exact location.These findings should provide a new perspective on the molecular mechanism of INH activation,which may have broad biomedical and toxicological significance for future research for more efficient or less resistant hydrazide anti-tuberculosis drugs.