Pituitary function has been shown to be regulated by an increasing number of intrapituitary factors, including cytokines. Here we show that the important cytokine TNF-α activates prolactin gene transcription in pituitary GH3 cells stably expressing luciferase under control of 5 kb of the human prolactin promoter. Similar regulation of the endogenous rat prolactin gene by TNF-α in GH3 cells was confirmed using real-time PCR. Luminescence microscopy revealed heterogeneous dynamic response patterns of promoter activity in individual cells. In GH3 cells treated with TNF-α, Western blot analysis showed rapid inhibitory protein κB (IκBα) degradation and phosphorylation of p65. Confocal microscopy of cells expressing fluorescence-labeled p65 and IκBα fusion proteins showed transient cytoplasmic-nuclear translocation and subsequent oscillations in p65 localization and confirmed IκBα degradation. This was associated with increased nuclear factor κB (NF-κB)-mediated transcription from an NF-κB-responsive luciferase reporter construct. Disruption of NF-κB signaling by expression of dominant-negative variants of IκB kinases or truncated IκBα abolished TNF-α activation of the prolactin promoter, suggesting that this effect was mediated by NF-κB. TNF-α signaling was found to interact with other endocrine signals to regulate prolactin gene expression and is likely to be a major paracrine modulator of lactotroph function. Copyright © 2006 by The Endocrine Society.