We investigated whether or not a 50 kDa glycoprotein might play an important role in protein synthesis-independent thermotolerance development in C H O cells. When cells were heated for 10 min at 45.5"C, they became thermotolerant to a heat treatment at 45.5"C administered 12 hr later. The thermotolerance ratio at 1 Op3 isosurvival was 4.4. The cellular heat shock response leads to enhanced glycosylation of a 50 kDa protein. The glycosylation of proteins including a 50 kDa glycoprotein was inhibited by treatment with various concentrations of tunicamycin (0.2-2 pghl). The development of thermotolerance was not affected by treatment with tunicamycin after the initial heat treatment, although 2 p g h l tunicamycin inhibited glycosylation by 95%. However, inhibiting protein synthesis with cycloheximide (1 0 kg/ml) after the initial heat treatment partially inhibited the development of thermotolerance. Nevertheless, there was no further reduction of thermotolerance development by treatment with a combination of 2 p d m l tunicamycin and 10 pgiml cycloheximide. These data suggest that development of thermotolerance, especially protein synthesis-independent thermotolerance, is not correlated with increased glycosylation of the 50 kDa protein.