Leishmania donovani, the causative agent of visceral leishmaniases, is an intracellular pathogen which proliferates within the host macrophages. Analysis of the thiol composition of L. donovani by means of the thiol-specific reagent, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, indicated that this organism produces substantial amounts of ovothiol A. This observation was further substantiated by HPLC of extracts of L. donovani after derivatization with bromobimane. L. donovani extracts contained a thiol, the bimane derivative of which had identical retention time and fluorescence quenching to a thiol from Crithidia fasciculata, which had previously been identified as ovothiol A. By comparison, the intracellular bacterial pathogen, Mycobacterium bovis, contained only one major low-molecular-mass thiol, which was assigned the trivial name mycothiol. The structure of the bimane derivative of mycothiol was solved by a combination of one- and two-dimensional 1H and 13C NMR spectroscopy. Spatial relationships in the molecule were further refined by NOE experiments and allowed identification of mycothiol as 1-D-myo-inositol-2-(N-acetyl-L-cysteinyl)amino-2-deoxy-alpha-D-glucopyra noside. This assignment was confirmed by positive-ion fast-atom-bombardment mass spectrometry which gave m/z = 677.6 Da and a sodiated species at 699.6 Da. Analysis of the dansylated hydrolysis products of performic-acid-oxidized mycothiol indicated the presence of 0.85 mol glucosamine and 1.02 mol cysteic acid/mol sulfhydryl groups. Crude extracts of M. bovis contained an enzyme which catalysed the NAD(P)H2-dependent reduction of mycothiol disulfide to the free thiol. Analysis of perchloric acid extracts of Mycobacterium tuberculosis H37RV indicated the presence of a thiol which comigrated with mycothiol, both as the free thiol and as the 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and bimane derivatives, on reverse-phase HPLC. The significance of these findings in terms of the evasion of the host defense mechanisms by leishmania parasites and mycobacteria is considered.