The six-degrees-of-freedom elastic behavior of spinal motion segments can be approximated by a stiffness matrix. A method is described to measure this stiffness matrix directly with the motion segment held under physiological conditions of axial preload and in an isotonic fluid bath by measuring the forces and moments associated with each of the six orthogonal translations and rotations. The stiffness matrix was obtained from the load-displacement measurements by linear least squares assuming a symmetric matrix. Results from a pig lumbar spinal motion segment in an isotonic bath, with and without a 500 N axial preload, showed a large stiffening effect with axial preload.