Pituitary function has been shown to be regulated by an increasing number of intra-pituitary factors including cytokines. Here we show that the important cytokine TNF- 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- 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-, Western blot analysis showed rapid IB degradation and phosphorylation of p65. Confocal microscopy of cells expressing fluorescence labeled p65 and IB fusion proteins showed transient cytoplasmic-nuclear translocation and subsequent oscillations in p65 localization and confirmed IB degradation. This was associated with increased NF-B mediated transcription from an NF-B responsive luciferase reporter construct. Disruption of NF-B signaling by expression of dominant negative variants of IKKs or truncated IB abolished TNF- activation of the prolactin promoter, suggesting that this effect was mediated by NF-B. TNF- 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.