When a clone of Chinese hamster ovary (CHO) cells transfected with a plasmid containing a luciferase reporter gene under the control of the human heat shock protein (hsp) 70 gene promoter was treated with cycloheximide during heat exposure at 42 and 43°C for 15 to 100 minutes and then incubated at 37°C after removal of cycloheximide, reporter gene expression was suppressed by the protein synthesis inhibitor only at small heat shock doses (i.e., heat shock of less than 40 minutes at 42°C and 15 minutes at 43°C). A similar stress dose-dependent suppression of reporter gene expression by cycloheximide was also demonstrated by treatment with sodium arsenite at 37°C. However, dexamethasone-dependent reporter gene expression in a different CHO clone was not inhibited after the inducer treatment for different times in the presence of cycloheximide. In addition, synthesis of most cellular proteins (except for hsp) was not affected after heat shock treatment with cycloheximide. The results suggested that the cycloheximide inhibition of gene expression is specific to hsp gene expression induced by limited stress doses. Furthermore, a prior 42°C heat shock treatment for 30 minutes induced a decreased responsiveness (tolerance) to a second 42°C heat treatment for hsp gene expression, but tolerance did not develop in cells exposed to the first heat shock in the presence of cycloheximide. These results confirm previous findings that induction of hsp gene expression by stress is balanced by the severity of stress and rate of protein synthesis. They also support the proposed model of autoregulation of hsp gene expression by levels of free hsp70.