To investigate the relationships between the loci expressing functions of estrogen receptor (ER)alpha and that of ERbeta, we analyzed the subnuclear distribution of ERalpha and ERbeta in response to ligand in single living cells using fusion proteins labeled with different spectral variants of green fluorescent protein. Upon activation with ligand treatment, fluorescent protein-tagged (FP)-ERbeta redistributed from a diffuse to discrete pattern within the nucleus, showing a similar time course as FP-ERalpha, and colocalized with FP-ERalpha in the same discrete cluster. Analysis using deletion mutants of ERalpha suggested that the ligand-dependent redistribution of ERalpha might occur through a large part of the receptor including at least the latter part of activation function (AF)-1, the DNA binding domain, nuclear matrix binding domain, and AF-2/ligand binding domain. In addition, a single AF-1 region within ERalpha homodimer, or a single DNA binding domain as well as AF-1 region within the ERalpha/ERbeta heterodimer, could be sufficient for the cluster formation. More than half of the discrete clusters of FP-ERalpha and FP-ERbeta were colocalized with hyperacetylated histone H4 and a component of the chromatin remodeling complex, Brg-1, indicating that ERs clusters might be involved in structural changes of chromatin.