Heat shock protein 27 (Hsp27) is a ubiquitously expressed member of the heat shock protein family that has been implicated in various biological functions including the response to heat shock, oxidative stress, and cytokine treatment. Previous studies have demonstrated that heat shock proteins are involved in regulating signal transduction pathways including the NF-kappa B pathway. In this study, we demonstrated that Hsp27 associates with the I kappa B kinase (IKK) complex and that this interaction was stimulated by tumor necrosis factor alpha treatment. Phosphorylation of Hsp27 by the kinase mitogen-activated protein kinase-activated protein kinase 2, a downstream substrate of the mitogen-activated protein kinase p38, enhanced the association of Hsp27 with IKK beta to result in decreased IKK activity. Consistent with these observations, treatment of cells with a p38 inhibitor reduced the association of Hsp27 with IKK beta and thus resulted in increased IKK activity. These studies indicate that Hsp27 plays a negative role in down-regulating IKK signaling by reducing its activity following tumor necrosis factor alpha stimulation.