Glucocorticoid hormone-dependent maturation of the mouse mammary tumor virus (MMTV) phosphorylated polyprotein (Pr74) allows experimental access to certain posttranslational regulatory circuits under steroid control in M1.54 cells, an MMTV-infected rat hepatoma cell line. Pulse-chase experiments revealed that [35S]methionine-labeled Pr74 synthesized in uninduced cells could be converted posttranslationally into p24, a stable phosphorylated maturation product, only after 4 h of exposure to 1 microM dexamethasone, a synthetic glucocorticoid. This regulated processing could be prevented by prior exposure, during the chase period, to inhibitors of RNA (actinomycin D) or protein (cycloheximide or puromycin) synthesis. Moreover, half-maximal production of p24 occurred at 10 nM dexamethasone, a concentration that approximated half-maximal receptor binding and stimulation of MMTV transcript synthesis. Kinetic, hormonal, and genetic evidence suggest that p24 expression did not require or result from the overall glucocorticoid-dependent increase in polyprotein concentration. First, 20 h after dexamethasone withdrawal, Pr74 maturation was completely deinduced, whereas the absolute level of this MMTV precursor remained 10-fold over its basal level. Second, progesterone, which competes with dexamethasone for receptor binding, facilitated the regulated production of p24 but prevented the steroid-mediated accumulation of functional MMTV mRNA. Lastly, certain glucocorticoid-responsive variants, derived from M1.54 cells by resistance to complement cytolysis, expressed p24 in the presence or absence of glucocorticoid-induced levels of Pr74. Taken together, our results suggest that the glucocorticoid-regulated maturation of MMTV phosphopolyproteins resulted from an independent hormone response that required normal receptor function and de novo RNA and protein synthesis.