Treatment of MCF-7 cells, a human mammary adenocarcinoma cell line, with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (10-7 M) was associated with a time-dependent reduction in the level of estrogen receptor (ER) mRNA (half-life approximately 3 h). In the presence of the RNA synthesis inhibitor actinomycin D [5.0 micrograms/ml (4.0 microM)], half-life of ER mRNA was much longer (approximately 12 h). Furthermore, the TPA-dependent down-regulation of ER mRNA was abolished by actinomycin D. Similar effects were observed with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (150 microM), an inhibitor of RNA polymerase. Inhibition of protein synthesis by cycloheximide (50 microM) or puromycin [50 micrograms/ml (92 microM)] did not alter the steady state level of ER mRNA during a period of 10-12 h. Furthermore, these inhibitors of protein synthesis did not prevent the down-regulation of ER mRNA in the presence of TPA. Our studies show that degradation of ER mRNA by TPA in MCF-7 cells is dependent on ongoing RNA synthesis but not on protein synthesis. This indicates that an RNA molecule with rapid turnover, which does not require translation, might be involved in the TPA-dependent ER mRNA decay.