To study the roles of nonmuscle myosin II (NM-II) during invasive cell migration, microfluidic migration chambers have been designed and fabricated using photo- and soft-lithography microfabrication techniques. The chamber consists of two channels separated by a vertical barrier with multiple bays of pores with widths varying from 6 microm to 16 microm, and lengths varying from 25 microm to 50 microm. The cells are plated in the channel on one side of the barrier while a chemoattractant is flowed through the channel on the other side of the barrier. In these chambers, cells can be observed with transmitted light or fluorescence optics while they chemotax through various sized pores that impose differential mechanical resistance to transmigration. As an initial test of this device, we compared breast-cancer cell chemotactic transmigration through different pore sizes with and without inhibition of NM-II. Two distinct rates were observed as cells attempted to pull their nucleus through the smaller pores, and the faster nuclear transit mode was critically dependent on NM-II motor activity. The ability to monitor cells as they chemotax through pores of different dimensions within a single experimental system provides novel information on how pore size affects cell morphology and migration rate, providing a dramatic improvement of imaging potential relative to other in vitro transmigration systems such as Boyden chambers.