There are two separate estrogen receptors (ERs), ERα and ERβ. The ERβ gene is variably spliced, and in some cases variant expression is high. Besides the full-length ERβ (equivalent to ERβ), splice variants can encode proteins bearing an insert within the ligand-binding domain (β32), a deletion of exon 3 (ERβ1δ3) disrupting the DNA-binding domain, or both (ERβ2δ3). Here we examine the intracellular localization and transcriptional properties of each of the ERβ splice variants heterologously expressed in cultured cells. In accordance with ERα, ERβ1 and ERβ2 are both distributed in a reticular pattern within the nucleus after exposure to ligand. In contrast, ERβ1δ3 and ERβ2δ3 localize to discrete spots within the nucleus in the presence of ER agonists. In the presence of ER antagonists, the δ3 variants are distributed diffusely within the nucleus. We also show that the spots are stable nuclear structures to which the δ3 variants localize in a ligand-dependent manner. Coactivator proteins of ER colocalize with δ3 variants in the spots in the presence of agonists. The δ3 variants of ERβ can activate luciferase reporter constructs containing an activator protein complex-1 site, but not an estrogen response element (ERE). These data suggest that without an intact DNA-binding domain, ERβ is functionally altered, allowing localization to discrete nuclear spots and activation from activator protein-1-containing reporter genes.