To examine activity of estrogen receptor-beta (ERbeta) independently of estrogen receptor-alpha (ERalpha), retrovirus-mediated gene transfer was used to insert rat ERbeta into a rat fibroblast cell line (rat-1) that does not ordinarily express ER. Stable expression of ERbeta in rat-1 cells was validated and then characterized by reverse-transcription polymerase chain-reaction (RT-PCR) analysis to examine the effects of estradiol (E2) treatment on expression of specific target mRNAs. Results were compared with rat-1 cells and a previously constructed rat-1 + ERalpha cell line. Progesterone receptor mRNA was not detected in rat-1 cells and was induced by E2 in both rat-1 + ERalpha and rat-1 + ERbeta cells. Treatment with E2 resulted in an increased rate of cell proliferation (P < 0.05) in rat-1 + ERalpha cells, but not in rat-1 or rat-1 + ERbeta cells. Data confirm studies using transient ER expression demonstrating that ERalpha and ERbeta have both discrete and overlapping activity within the same cell type in the presence of the same ligand.