Ribosome production is closely coupled to the proliferative state of the cell and responds rapidly to changes in the demand for protein synthesis caused by various physiological stimuli. Control of ribosome biogenesis is accomplished by regulating the synthesis of the various precursor molecules that are assembled into mature ribosomes. To determine the mechanisms by which insulin and dexamethasone, a glucocorticoid analogue, regulate ribosome precursor synthesis, the effect of these hormones on transcription and translation of ribosomal protein L32 (rpL32) was examined. Insulin treatment of serum starved C2C12 myoblasts resulted in a 3-fold increase in the rate of rpL32 gene transcription within 4 hours. The promoter of the rpL32 gene contains four discrete elements ([beta], [gamma], [delta] 1, [delta]2) characterized by their requirement for efficient transcription and ability to bind nuclear factors. Following insulin treatment, an increase in 0 factor binding was observed within 2 hours, while binding activities of the [delta] and [gamma] factors remained unchanged. Furthermore, insulin treatment resulted in a rapid increase in rpL32 mRNA translation as demonstrated by selective redistribution of rpL32 mRNA into polysome pools of stimulated myoblasts. Insulin mediated stimulation of rpL32 transcription and translation was suppressed predominantly by wortmannin and rapamycin indicating the importance of PI 3-kinase and FRAP/mTOR pathways in the insulin effect. Taken together these results indicate that insulin increases rpL32 production by both transcriptional and translational mechanisms. Incubation of L6 myoblasts with dexamethasone for 12 hours resulted in a 2.5-fold increase in the rate of rpL32 gene transcription with corresponding increases in rpL32 mRNA levels. In concert with the rise in transcription, binding of the 5 factor increased during the same time period without significant effect on [beta] and [gamma] factor binding. The presence of the glucocorticoid antagonist RU38486 reversed the stimulatory effect of dexamethasone on rpL32 gene transcription with a parallel reduction in binding activity of the [delta] factor to levels observed in untreated cells. These results suggest that dexamethasone enhances rpL32 gene transcription through glucocorticoid receptor mediated changes in the activity of the [delta] factor. Therefore, although both insulin and dexamethasone stimulate rpL32 synthesis, they mediate their effects through different mechanisms.