Microbacterium sp. M 874 produced a glyceroglycolipid, di-O-12-methyl-tetradecanoyl-3-O-β-D-galactopyranosyl-sn-glycerol, at about the 50 μM level. Though the strain was highly resistant to tertiary-butyl hydroperoxide (tBHP) in a glycolipid-productive medium, the resistance was reduced in a nonproductive medium. Exogenous addition of the glycolipid to the nonproductive culture restored the resistance. This addition also increased the resistance to heat, ethanol, and 4-chloro-l-naphthol, in which oxygen radicals might participate. The parallel relationship found in strain M 874 mutants between glycolipid productivity and resistance to tBHP or heat suggested that the resistance was mainly caused by the glycolipid. On addition of the glycolipid to a glycolipid-nonproductive culture, it was immediately incorporated into the cells and functioned as an antioxygen radical reagent. Thereafter, its intracellular level remained largely unchanged for at least 5 h, even in the presence of tBHP, and its activity was maintained. The glycolipid at 142 μM was sufficient to prevent the cytotoxicity induced by 88.9mM tBHP. The glycolipid production was not induced by pretreatment with a low level of tBHP or a sublethal heat shock. In brief, the glycolipid might play an essential role in the prevention of damage by oxygen radicals in the glycolipid-producing bacterium.