Since cell migration plays critical roles in development and homeostasis of the body, artificial control of cell migration would be promising for the treatment of various diseases related to migration. To this end, we previously developed single-chain Fv (scFv)/receptor chimeras, named signalobodies, which can control cell fates via a specific antigen that is different from natural cytokines. Although a conventional chemotaxis chamber assay revealed that several signalobodies based on receptor tyrosine kinases transduced antigen-dependent migration signals, we have never performed direct observation of the cells to obtain more information on overall properties of cell motility and migration. In this study, we utilized murine pro-B Ba/F3 cells expressing either a scFv-Fms or scFv-Mpl signalobody, and compared their migratory characteristics. We employed a lipid-polyethylene glycol conjugate to softly immobilize the suspension cells on a slide, which facilitated direct observation of chemokinetic activity of the cells. Consequently, both cells markedly exhibited chemokinesis in response to a specific antigen. In addition, the cells were subjected to a stable antigen-concentration gradient to observe horizontal directional cell migration in real time. The results showed that the cells expressing scFv-Fms underwent directional migration toward a positive antigen-concentration gradient. Taken together, we successfully demonstrated antigen-responsive regulation of cell motility and migration via the signalobodies. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:411-417, 2014