Nuclear receptors initiate transcription, interact with regulatory proteins, and are influenced by hormones, drugs, and pollutants. Herein, we discover ligand-specific mobility patterns of human estrogen receptor-alpha (ER) in living cells using diffusion-time distribution analysis (DDA). This novel method, based on fluorescence correlation spectroscopy (FCS), is especially suited to unraveling multiple protein interactions in vivo at native expression levels. We found that ER forms a limited number of distinct complexes with a varying population by dynamic interaction with other nuclear components. Dose-response curves of different ligands could be obtained for each receptor interaction. The potential to identify interacting proteins was demonstrated by comparing DDA of the ER cofactor SRC-3 attached to yellow fluorescent protein (YFP) with those of YFP-ER. Our findings open up new routes to elucidating transcription regulation and to detecting and distinguishing pharmacologically and toxicologically active compounds in vivo. Moreover, DDA provides a general approach to monitoring biochemical networks in individual living cells.