The mycolic acid compositions of Nocardia rubra and related bacteria grown in media containing different concentrations of antituberculous isonicotinic acid hydrazide (INH) were determined in detail by gas chromatography-mass spectrometry. On the basis of molecular species composition, average carbon numbers of mycolic acids were calculated. In Nocardia rubra, N. lutea and Rhodococcus rhodochrous IFO-13161, the ratio of mycolic to non-mycolic fatty acids and the average carbon numbers of mycolic acids were decreased at the INH concentrations of higher than 1 microgram/ml, paralleling with the significant inhibition of growth. In above three species the synthesis of longer chain mycolic acids (longer than C44 or C46 ) was inhibited more significantly than shorter homologues such as C38 or C40 . In contrast, neither growth inhibition nor change in corynomycolic acid composition was observed in Corynebacteria xerosis and Rhodococcus rhodochrous IFO-13165 at the concentration region of INH up to 100 micrograms/ml. The direct mass fragmentographic analysis of the trimethylsilylated (TMS) derivatives of mycolic acid methyl esters, monitoring [M-15] ions of individual molecular species, revealed that the chain shortening of total mycolic acid molecule by INH occurred more greatly in more highly unsaturated subclasses than in less unsaturated subclasses. Furthermore, mass fragmentographic analysis, monitoring fragment ions (A) and (B), due to straight chain and branched chain alkyl units, respectively, demonstrated the inhibition of mycolic acids was not attributed to the shortening of alpha-alkyl chain, but to the inhibition of chain elongation of C28 to C32 straight chain meromycolic acids. It was also indicated the amounts of trehalose mono- and di- mycolate (cord factor) decreased significantly with the addition of INH (1 to 20 micrograms/ml) in the above strains. From the results obtained above, INH appeared to inhibit the synthesis of mycolic acids longer than C44 or C46 specifically by inhibiting chain elongation or desaturation of precursor long chain fatty acids longer than C28 or C30.