The NF-kappaB pathway is important in the control of the immune and inflammatory response. One of the critical events in the activation of this pathway is the stimulation of the IkappaB kinases (IKKs) by cytokines such as tumor necrosis factor-alpha and interleukin-1. Although the mechanisms that modulate IKK activation have been studied in detail, much less is known about the processes that down-regulate its activity following cytokine treatment. In this study, we utilized biochemical fractionation and mass spectrometry to demonstrate that protein phosphatase 2Cbeta (PP2Cbeta) can associate with the IKK complex. PP2Cbeta association with the IKK complex led to the dephosphorylation of IKKbeta and decreased its kinase activity. The binding of PP2Cbeta to IKKbeta was decreased at early times post-tumor necrosis factor-alpha treatment and was restored at later times following treatment with this cytokine. Experiments utilizing siRNA directed against PP2Cbeta demonstrated an in vivo role for this phosphatase in decreasing IKK activity at late times following cytokine treatment. These studies are consistent with the ability of PP2Cbeta to down-regulate cytokine-induced NF-kappaB activation by altering IKK activity.