Our previous studies have shown that polyamines are essential for early mucosal restitution in vivo and cell migration in vitro. The current study determines whether cytoskeleton is involved in the process requiring polyamines for the stimulation of cell migration. Treatment with α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis, for 4 days totally inhibited ODC activity and depleted intracellular polyamines in the intestinal epithelial cells (IEC-6) derived from rat small intestinal crypt cells. Polyamine deficiency resulted in reorganization of F-actin in migrating cells but had no effect on the concentrations of filamentous actin and β-actin mRNA. The actin cortex was greatly increased in density and lamellipodia were less extensive. In contrast, non-muscle myosin I and II levels in DFMO-treated cells were decreased by 70 and 75%, respectively, and stress fibres were sparse or absent. The most striking feature of DFMO-treated cells was the appearance of many small punctate foci of myosin II in the cell interior. Migration of DFMO-treated cells was reduced by 80%. In the presence of DFMO, exogenous polyamine not only returned cytoskeleton levels and distribution towards normal but also restored cell migration to control levels. These results indicate that actin and myosins have a role in polyamine-dependent epithelial cell migration and may be part of the mechanism that requires polyamines for early mucosal restitution.