Glucocorticoid effects on insulin biosynthesis and secretion have been controversial. To define whether glucocorticoids affect insulin biosynthesis in vitro, the actions of dexamethasone on insulin gene expression were examined using the HIT cell line, a transformed clonal line of hamster beta-cells. Dexamethasone induced a dose-dependent decrease in steady-state insulin messenger RNA (mRNA) levels, which was prevented by adding an excess of RU 486, a competitive inhibitor for the binding to the glucocorticoid receptor. Inhibition was not observed before 6 h of dexamethasone treatment and was maximal at 24 h. To further assess the molecular mechanisms of the dexamethasone-induced decreases in insulin mRNA levels, we investigated whether transcription of the insulin gene was affected. Run-on assays revealed that transcription rates were not changed by glucocorticoids. Inhibition of RNA and protein synthesis by actinomycin D and cycloheximide, respectively, completely abolished the dexamethasone effects, whereas actinomycin D added 9 h after dexamethasone had no effect on insulin mRNA levels. The present results demonstrate that glucocorticoids can acutely inhibit insulin biosynthesis by destabilizing insulin mRNA; this effect requires the transcriptional activation of a gene encoding a protein responsible for the accelerated disappearance of insulin mRNA.