We examined the early effects of ethambutol on the synthesis of trehalose monomycolate, trehalose dimycolate, and free mycolic acid in actively growing cells of Mycobacterium smegmatis. At about 1 min after the addition of 3.0 micrograms of ethambutol per ml, the cellular level of trehalose monomycolate began to increase over the control culture. This was followed 8 to 12 min later by the cellular increases in free mycolic acid and trehalose dimycolate over the control culture and the inhibition of incorporation of mycolic acid into the cell wall. Exposure of M. smegmatis to ethambutol for more than 30 min caused all of these lipids to leak out of the cells more rapidly than in the control cells. The mechanism by which ethambutol initiates these events is unknown, but these early imbalances in lipid synthesis may be responsible for the lethal action of this drug.