When an actively growing culture of the H37Ra strain of Mycobacterium tuberculosis was exposed to isoniazid at a concentration of 0.5 mug/ml, the cells began to lose their ability to synthesize mycolic acids immediately. After 60 min, the cells had completely lost this ability. The synthesis of the three mycolate components-alpha-mycolate, methoxymycolate, and beta-mycolate-was inhibited. The viability of the isoniazid-treated cells was unaffected up to about 60 min of exposure, after which time there was a gradual decline in the viability to about 18% after 180 min. Correspondingly, growth of the drug-treated cells slowed down and stopped after 24 hr. The inhibition of the synthesis of mycolic acids was reversible if the drug was removed before the loss of viability set in. Incubation of the viable cells in the absence of the drug for 24 hr restored the mycolate synthesis. These results strongly suggest that the inhibition of the synthesis of the mycolic acids is closely associated with the primary mechanism of action of isoniazid on the tubercle bacilli. The sequence of events which leads to the loss of viability of cells exposed to isoniazid is described.