We have previously developed a temperature-responsive cell culture surface by grafting poly(N-isopropylacrylamide) that changes its surface hydrophobicity in response to temperature. While this surface shows similar hydrophobicity to that of commercial polystyrene cell culture surfaces and facilitates cell adhesion and proliferation at 37 degrees C, grafted polymer becomes hydrophilic below 32 degrees C and releases spread cultured cells without trypsin. Temperature-regulated cell detachment requires cell metabolic activity requiring ATP consumption, signal transduction, and cytoskeleton reorganziation. Precoating these surfaces with fibronectin (FN) improves spreading of less adhesive cultured hepatocytes and reducing culture temperature releases cultured cells from FN-adsorbed grafted surfaces. Immunostaining with anti-FN antibody revealed that only FN located beneath cultured cells is removed from culture surfaces after reducing temperature. FN adsorbed to surface areas lacking direct cell attachment remained surface-bound after reducing temperature. A novel concept of active cell detachment is also discussed.