Epidemiological data have implicated perturbations in the regulation of NF-B activity to diseases that affect a large number of Americans today. Specifically, chronic activation of genes involved in the inflammatory response is associated with the progression of and complications in diabetes, arthritis, atherosclerosis, and cancer. Insight into the mechanisms governing the regulation of NF-B transcriptional activity will provide the molecular link between NF-B and these pathological states. SIMPL (signaling molecule that associates with mouse pelle-like kinase) is a component of a signaling pathway through which tumor necrosis factor-alpha (TNF) induces NF-B controlled gene transcription. SIMPL interacts with the nuclear pool of the NF-B subunit, p65, in a TNF dependent manner to enhance p65 dependent gene transcription. How SIMPL activity is regulated is unknown. Under basal as well as TNF stimulated conditions, SIMPL phosphopeptides were identified. SIMPL mutants lacking sites that are phosphorylated under basal conditions diminished p65 trans-activation activity but had no effect upon SIMPL nuclear localization. SIMPL mutants lacking sites of TNF induced phosphorylation impaired nuclear localization and prevented TNF induced p65 trans-activation activity. Together these studies reveal that phosphorylation of the SIMPL protein plays a critical role in SIMPL regulation by affecting both SIMPL subcellular localization as well as the p65 co-activator function of SIMPL.