Incubation of MCF-7 cells with estradiol (E2) down-regulates estrogen receptor (ER) resulting in a progressive reduction of the capacity of cells to concentrate selectively [3H]E2. Scatchard plot analysis failed to detect any transformation of residual receptors into peptides of lower binding affinity. [3H]Estrone gave an identical ER disappearance pattern with an ER half-life comprised between 2 and 3 h. A similar value was established by incubating the cells with [3H]tamoxifenaziridine ([3H]TAZ) for 1 h before the addition of excessive unlabeled E2 which induced ER-down regulation and impeded any further labeling of the residual receptors. Submission of the [3H]TAZ labeled cell extracts to SDS-PAGE revealed no progressive emergence of low molecular weight cleavage products of the receptor (< 67 kDa). Two inhibitors of protein kinases, H-7 at 40 microM and H-89 at 20 microM, failed to block the E2-induced ER down-regulation. On the contrary, the protein phosphatases 1 and 2A inhibitor, okadaic acid, was effective with concentrations higher than 0.1 microM indicating that a dephosphorylation mechanism was involved in this phenomenon. Cycloheximide (CHX) also significantly reduced the receptor decrease at concentrations higher than 1 microM. G-C specific intercalating agents [actinomycin D (AMD) and chromomycin A3 at 1 microM] also prevented ER disappearance; ethidium bromide (EB) and quinacrine were ineffective. AMD and CHX operated immediately after their addition to the medium indicating an inhibitory action on the synthesis of an RNA and/or a peptide with high turnover rate involved in ER decline. Moreover, AMD produced its suppressive effects under conditions impeding any labeling of newly synthetized receptors (i.e. [3H]TAZ with an excess of unlabeled E2) rejecting the possibility of an increasing ER production which may partially hamper its disappearance. Finally, E2-induced ER mRNA down-regulation was similarly abolished by AMD while EB and CHX were devoid of effect.