We report a mammalian-based promoter chromosomal array system developed for single-cell studies of transcription-factor function. Designed after the prolactin promoter-enhancer, it allows for the direct visualization of estrogen receptor alpha (ERalpha) and/or Pit-1 interactions at a physiologically regulated transcription locus. ERalpha- and ligand-dependent cofactor recruitment, large-scale chromatin modifications and transcriptional activity identified a distinct fingerprint of responses for each condition. Ligand-dependent transcription (more than threefold activation compared with vehicle, or complete repression by mRNA fluorescent in situ hybridization) at the array correlated with its state of condensation, which was assayed using a novel high throughput microscopy approach. In support of the nuclear receptor hit-and-run model, photobleaching studies provided direct evidence of very transient ER-array interactions, and revealed ligand-dependent changes in k(off). ERalpha-truncation mutants indicated that helix-12 and interactions with co-regulators influenced both large-scale chromatin modeling and photobleaching recovery times. These data also showed that the ERalpha DNA-binding domain was insufficient for array targeting. Collectively, quantitative observations from this physiologically relevant biosensor suggest stochastic-based dynamics influence gene regulation at the promoter level.