An amino-terminal fusion of the human estrogen receptor alpha (ER) with human O6-alkylguanine-DNA alkyltransferase (AGT) enabled the observation and distinction of consecutively expressed ER populations by sequential pulse labeling of the AGT tag with different fluorescent O(6)-alkylguanine derivatives in live cells. The application of agonists and antagonists led to the characteristic speckled redistribution of fluorescent receptors in the nucleus as visualized by confocal microscopy. To investigate where newly synthesized receptors were localized in individual cells with respect to their older relatives in response to extracellular chemical signals, receptor expression was continued for 4 h and newly synthesized receptors were labeled with a new fluorophore spectrally distinct from the first probe. This strategy enabled a time-resolved analysis of the formation of ER-enriched protein complexes in distinct nucleoplasmic compartments. Such complexes represent important but hitherto uncharacterized macromolecular structures involved in ER function. Different, long-lasting effects were observed depending on the type of ligand. For example, 4 h after pulsed application of the partial antagonist 4-hydroxytamoxifen, the second receptor population exhibited a speckled pattern in the cell nucleus that overlapped with the first receptor population pattern. This novel finding suggests that the intranuclear positioning of receptor aggregates is not random but influenced in a ligand-dependent manner. The antagonist ICI 182,780 (7-alpha-[9-(4.4,5,5,5-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10)-triene-3,17-beta-diol), a potent drug used in cancer treatment, led to down-regulation of the first receptor population and newly expressed receptors accumulated in the cytoplasm. In contrast, the natural agonist 17beta-estradiol resulted in significantly shorter effects. Four hours after ligand application, newly expressed receptors were homogeneously distributed in the nucleus as in untreated control cells. We present the pulse labeling of AGT-ER fusion proteins with different fluorophores as a novel tool for investigating the functional regulation of nuclear receptors in individual cells.