We previously developed a cell separation method using a poly(N-isopropylacrylamide)-grafted polypropylene (PNIPAAm-g-PP) membrane containing an adsorbed monoclonal antibody (mAb). The purpose of this study is to elucidate the cell separation mechanism in detail and to design an optimal method. As the grafting yield of PNIPAAm increased, the level of the adsorption of IgG(1) and cell adhesion to the membrane decreased. After BSA was adsorbed to a PNIPAAm-g-PP membrane at 6 degrees C, where PNIPAAm was hydrophilic, a small amount of IgG(1) was adsorbed to the membrane at 37 degrees C, where PNIPAAm was hydrophobic. The desorption of the adsorbed IgG(1) was not enhanced even though temperature was reduced to 10 degrees C, where PNIPAAm was hydrophilic. These results indicate that the antibody adsorbed to the intact PP surface of the membrane predominantly contributes to the capture of target cells through the antigen-antibody reaction and that a thermoresponsive transition of PNIPAAm contributes to the detachment of the captured cells. The total number of cells recovered from a PNIPAAm-g-PP membrane containing the adsorbed mAb decreased as the grafting yield increased. A PNIPAAm-g-PP membrane with a 1.7% grafting yield containing adsorbed anti-human CD34 mAb enriched CD34-positive KG-1a cells to 85% from a 1:1 cell suspension of KG-1 a cells and CD34-negative Jurkat cells.