The two known forms of estrogen receptor (ER), α and β, exhibit differences in structure, affinity for certain ligands, and tissue distribution, suggesting differential roles. It is of interest from several perspectives to determine whether the two receptors elicit similar or differing responses within the same cell type in the presence of the same ligand. To evaluate roles of ER, we have examined responses to estrogen in a rat embryonic fibroblast cell line model, normally naive to ER, engineered to stably express ERα or ERβ. Rat1 ERα, Rat1 ERβ, and precursor Rat1 cell lines were treated with estradiol-17β (E₂; 1 nM) or an ethanol vehicle for 24 h. Total RNA was extracted, and cDNA generated and subjected to suppression subtractive hybridization (SSH), followed by differential screening using dot blot hybridization. In the presence of ERα, products were identified that represent classic responses to E₂, including markers for cell proliferation. In the presence of ERβ, an alternate transcription profile was observed, including upregulation of pro-alpha-2(I) collagen. These data support a model in which ERα and ERβ regulate unique subsets of downstream genes within a given cell type.