The combinatorial interactions of the homeodomain protein Pit-1 with transcription factors such as C/EBPalpha and with coregulatory proteins control the development of anterior pituitary cells. These cells fail to develop in individuals with mutations affecting the Pit-1 gene, resulting in combined pituitary hormone deficiency. The present work describes biochemical, live-cell imaging and in vivo kinetics studies that provide new insight into the dynamic network interactions inside the pituitary-cell nucleus. The findings reported here support a model where proteins move independently within the nuclear compartment and stochastically associate at specific intranuclear sites as part of metastable complexes. The probability that these proteins assemble in a particular region of the cell nucleus is related to the dominant chromatin-binding activities of the different protein partners, supporting the view that the repositioning of transcription factors and coregulators represents an important mechanism for directing changes in cell-specific gene expression. These studies also suggest an important link between the mislocalization of transcriptional complexes and disease processes.