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The morphology and function of rat GH3 pituitary cells are profoundly affected by estradiol-17beta (E2), presumably due to changes in the profile of gene expression. We recently reported that a major target of E2 in these cells is the ezrin gene, which encodes a cytoskeletal linker protein that forms a complex with ezrin/radixin/ moesin-binding protein 50 (EBP50) in some cell types. Other studies have shown that EBP50 levels are increased by E2 in human breast and uterine tissue. Thus, we examined whether ezrin and EBP50 expression is coordinately increased by E2 in GH3 cells in vitro and rat pituitary glands in vivo. Ezrin levels are repressed by the steroidal antiestrogen, ICI 182780, and this effect is abrogated by E2 and the ERalpha-specific agonist, PPT, in GH3 cells. In contrast, EBP50 levels remained constant during these treatments. Ezrin and EBP50 did not display extensive colocalization. Moreover, ezrin was not co-immunoprecipitated by an EBP50 antibody in parental GH3 cells or in GH3 cells stably overexpressing EBP50, but was co-immunoprecipitated with EBP50 in human breast MCF-7 cells. Disruption of the actin cytoskeleton of GH3 cells changed the distribution of ezrin within subcellular fractions, but had no effect on EBP50. Finally, in juvenile female rats, E2 injections increased ezrin expression in the pituitary and uterus, but increased EBP50 expression only in the uterus. These findings demonstrate tissue specificity in the formation of ezrin-EBP50 complexes and in the regulation of EBP50 expression in estrogen-responsive tissues.