The estrogen receptor (ER) α is a hormone-inducible transcription factor that has a pivotal physiological role. Intriguingly, a clear and undisputed physiological function of the recently described ERβ remains elusive, with the exception of the ovary where a cooperative role of ERα and ERβ has been demonstrated. We have, therefore, investigated whether endogenous ERs, in particular ERβ, act as ligand-inducible transcription factors in primary ovarian cells derived from wild-type, ERα or ERβ knockout mice. Granulosa-enriched cell fractions naturally expressing ERβ and thecal cell fractions that express ERα were analyzed in transactivation assays using the vitellogenin A2 consensus estrogen response element and potent ER agonists diethylstilbestrol and S-indenestrol A. We studied also the potency-selective ERβ agonist R-indenestrol A, the pure ERα agonist and ERβ antagonist R,R-diethyl-tetrahydrochrysene and the pure ERα agonist propylpyrazole-triol. Using ER subtype-specific physiological cell models and these ER subtype-specific structural probes, we analyzed trans-activation of ERα and ERβ. This analysis revealed that endogenously expressed ERβ is indeed functional as a transcription factor, that it responds to estrogens appropriately, and that the ligands used are true ER subtype-specific probes in primary ovarian cells. In conclusion, this study demonstrates that endogenously expressed ERβ is capable of regulating gene transcription independent of ERα.