The traction forces exerted by an adherent cell on a substrate have been studied only in the two-dimensions (2D) tangential to substrate surface (Txy). We developed a novel technique to measure the three-dimensional (3D) traction forces exerted by live bovine aortic endothelial cells (BAECs) on polyacrylamide deformable substrate. On 3D images acquired by confocal microscopy, displacements were determined with image-processing programs, and traction forces in tangential (XY) and normal (Z) directions were computed by finite element method (FEM). BAECs generated traction force in normal direction (Tz) with an order of magnitude comparable to Txy. Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign reversal between cell edge and nucleus edge. The method was evaluated regarding accuracy and precision of displacement measurements, effects of FE mesh size, displacement noises, and simple bootstrapping. These results provide new insights into cell-matrix interactions in terms of spatial and temporal variations in traction forces in 3D. This technique can be applied to study live cells to assess their biomechanical dynamics in conjunction with biochemical and functional activities, for investigating cellular functions in health and disease. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12195-009-0082-6) contains supplementary material, which is available to authorized users.