Epidermal growth factor (EGF) plays an important role in the protection and repair of gastrointestinal mucosa, and it has been shown to stimulate migration of a number of cell types. However, the signaling mechanisms that regulate growth factor-mediated migration are as yet undefined. We have previously shown that polyamines are required for proliferation and migration both in vivo and in a cultured intestinal epithelial cell (IEC-6) model. In this study, we employed EGF and stable cell lines expressing constitutively active (CA) and dominant negative (DN) MEK1 to study the role of growth factor signaling in control and polyamine depleted cells. Wounding of the IEC-6 monolayer induced robust and transient ERK activation, which was further enhanced by EGF. EGF stimulated migration in control and polyamine-depleted cells, but the degree of stimulation was significantly less in polyamine-depleted cells. Inhibition of MEK1 by U0126 inhibited basal as well as EGF-induced ERK activation and migration. Expression of CA- and DN-MEK had significant effects on F-actin structure. Transfection with CA-MEK increased stress fiber and lamellipodia formation, while transfection of cells with DN-MEK- showed loss of stress fibers and normal actin cytoskeletal structure compared to cells transfected with vector. Unlike EGF, transfection with CA-MEK significantly increased migration of both control and polyamine-depleted cells equally indicating that sustained MEK activation is sufficient to circumvent the requirement for polyamines in the migration of IEC-6 cells. The most important and significant finding in this study was that polyamine depletion caused localization of Rac1 and RhoA to the nuclear as well as perinuclear regions. Interestingly, Transfection with CA-MEK completely reversed the sub-cellular distribution of Rac1 and RhoA proteins in polyamine-depleted cells. Polyamine depletion increased Rac1 levels in the nuclear fraction and decreased it in the cytoplasmic and membrane fractions of vector-transfected cells. Transfection with CA-MEK prevented accumulation of Rac1 in the nucleus. Polyamine depletion significantly decreased Rac1 activity during 6h migration in vector-transfected cells. Cells transfected with CA-MEK had almost identical levels of activated Rac1 in all three groups. These results suggest that polyamine depletion prevents activation of Rac1 and RhoA by sequestering them to the nucleus, and that expression of constitutively active MEK reverses this effect, creating the cellular localization required for activation.