The bending stiffness of a phospholipid bilayer (kc) was measured by forming thin bilayer cylinders (tethers) from giant phospholipid vesicles. Based on the balance of forces, the tether force was expeeted to be proportional to the square root of the membrane tension, with a constant of proportionality containingk>c. The membrane tension was controlled via the aspiration pressure in a micropipette used to hold the vesicle. The force on the tether was generated by an electromagnet acting on a paramagnetic bead attached to the vesicle surface. The magnitude of the force was determined from measurements on the magnet current which was adjusted to maintain the position of the bead. Measurements were performed on vesicles composed of stearoyl-oleoyl-phosphatidylcholine plus 5% (by mole) biotinylated phosphatidylethanolamine to mediate adhesion to streptavidin-coated beads. From each vesicle, tethers were formed repeatedly at different values of the membrane tension. The expected relationship between membrane tension and tether force was observed. The mean value ofkc for 10 different vesicles was 1.17×10⁻¹⁹ J (SD=0.08×10⁻¹⁹ J). The precision of these data demonstrates the reliability of this approach, which avoids uncertainties of interpretation and measurement that may be associated with other methods for determiningkc.