Epidermal cell migration is a key factor in wound healing responses, regulated by the F-actin-myosin II systems. Previous reports have established the importance of non-muscle myosin II (NMII) in regulating cell migration. However, the role of NMII in primary human keratinocytes has not been investigated. In this study we used a microfabrication-based two-dimensional migration assay to examine the role of NMII in keratinocyte migration. We developed confluent cell islands of various sizes (0.025 - 0.25 mm(2)) and quantified migration as Fold Increase in island area over time. We report here that NMII was expressed and activated in migrating keratinocytes. Inhibition of NMIIA motor activity with blebbistatin increased migration significantly in all cell island sizes in six hours compared to control. Inhibition of Rho-kinase by Y-27632 did not alter migration while inhibition of myosin light chain kinase by ML-7 suppressed migration significantly in six hours. Both blebbistatin and Y-27632 induced formation of large membrane ruffles and elongated tails. In contrast, ML-7 blocked cell spreading, resulting in a rounded morphology. Taken together, these data suggest that NMIIA decreases migration in keratinocytes, but the mechanism may be differentially regulated by upstream kinases.