The effect of growth temperature on mycolic acid composition in eight strains of Mycobacterium smegmatis was investigated by gas chromatography/mass spectrometry. A change in growth temperature from 45 to 20°C caused a shift in the subclass and molecular species composition of mycolic acids. The relative amount of α'-mycolic acids to α-mycolic acids decreased, and that of hydroxy mycolic acids increased at lower temperatures. Moreover, the proportion of shorter-chain species of α-mycolic acids increased, and those of longer-chain species of α-mycolic and hydroxy mycolic acids decreased. This observation seems to be due to the changes of the chain length of meromycolates because the α-alkyl chain unit of mycolic acids was not affected. The ratio of odd to even carbon-numbered α-mycolates decreased as the growth temperature was lowered. In contrast, the molecular species composition of α'-mycolic acid was not influenced by the growth temperature.