Polyamines are essential for cell migration during early mucosal restitution after wounding in the gastrointestinal tract. Activity of voltage-gated K(+) channels (Kv) controls membrane potential (E(m)) that regulates cytoplasmic free Ca(2+) concentration ([Ca(2+)](cyt)) by governing the driving force for Ca(2+) influx. This study determined whether polyamines are required for the stimulation of cell migration by altering K(+) channel gene expression, E(m), and [Ca(2+)](cyt) in intestinal epithelial cells (IEC-6). The specific inhibitor of polyamine synthesis, alpha-difluoromethylornithine (DFMO, 5 mM), depleted cellular polyamines (putrescine, spermidine, and spermine), selectively inhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression, and resulted in membrane depolarization. Because IEC-6 cells did not express voltage-gated Ca(2+) channels, the depolarized E(m) in DFMO-treated cells decreased [Ca(2+)](cyt) as a result of reduced driving force for Ca(2+) influx through capacitative Ca(2+) entry. Migration was reduced by 80% in the polyamine-deficient cells. Exogenous spermidine not only reversed the effects of DFMO on Kv1.1 channel expression, E(m), and [Ca(2+)](cyt) but also restored cell migration to normal. Removal of extracellular Ca(2+) or blockade of Kv channels (by 4-aminopyridine, 1-5 mM) significantly inhibited normal cell migration and prevented the restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These results suggest that polyamine-dependent intestinal epithelial cell migration may be due partially to an increase of Kv1.1 channel expression. The subsequent membrane hyperpolarization raises [Ca(2+)](cyt) by increasing the driving force (the electrochemical gradient) for Ca(2+) influx and thus stimulates cell migration.