We have *analyzed ligand-dependent, subnuclear movements of the estrogen receptor-α (ERα) in terms of both spatial distribution and solubility partitioning. Using a transcriptionally active green fluorescent protein-ERα chimera (GFP-ERα), we find that 17β-estradiol (E₂) changes the normally diffuse nucleoplasmic pattern of GFP-ERα to a hyperspeckled distribution within 10–20 min. A similar reorganization occurs with the partial antagonist 4-hydroxytamoxifen; only a subtle effect was observed with the pure antagonist ICI 182,780. To examine the influence of ligand upon ERα association with nuclear structure, MCF-7 cells were extracted to reveal the nuclear matrix (NM). Addition of E₂, 4-hydroxytamoxifen, or ICI 182,780 causes ERα to partition with the NM-bound fraction on a similar time course (10–20 min) as the spatial reorganization suggesting that the two events are related. To determine the effects of E₂ on the redistribution and solubility of GFP-ERα, individual cells were directly examined during both hormone addition and NM extraction and showed that GFP-ERα movement and NM association were coincident. Colocalization experiments were performed with antibodies to identify sites of transcription (RNA pol IIo) and splicing domains (SRm160). Using E₂ treated MCF-7 cells, minor overlap was observed with transcription sites and a small amount of the total ERα pool. Experiments performed with bioluminescent derivatives of ERα and steroid receptor coactivator-1 (SRC-1) demonstrated both proteins colocalize to the same NM-bound foci in response to E₂ but not the antagonists tested. Deletion mutagenesis and in situ analyses indicate intranuclear colocalization requires a central SRC-1 domain containing LXXLL motifs. Collectively, our data suggest that ERα transcription function is dependent upon dynamic early events including intranuclear rearrangement, NM association, and SRC-1 interactions.