The effects of actin filaments (AFs) and microtubules (MTs) on quasi-in situ tensile properties and intracellular force balance were studied in cultured rat aortic smooth muscle cells (SMCs). A SMC cultured on substrates was held using a pair of micropipettes, gradually detached from the substrate while maintaining in situ cell shape and cytoskeletal integrity, and then stretched up to ~15% and unloaded three times at the rate of 1 µm every 5 seconds. The cell stiffness was ~20 nN per percent strain in the untreated case, and decreased by ~65% and ~30% following AF and MT disruption, respectively. MT augmentation did not affect cell stiffness significantly. The role of AFs and MTs in resisting cell stretching and shortening were assessed using the area retraction of the cell upon noninvasive detachment from thermoresponsive-gelatin-coated dishes. The retraction was ~40% in untreated cells, while it was less than 20% in AF-disrupted cells. The retraction increased by ~50% and decreased by ~30% following MT disruption and augmentation, respectively, suggesting that MTs resist intercellular tension generated by AFs. Three-dimensional measurements of cell morphology using confocal microscopy revealed that the cell volume remained unchanged following drug treatment. A concomitant increase in cell height and decrease in cell area was observed following AF disruption and MT augmentation. In contrast, MT disruption significantly reduced the cell height. These results indicate that both AFs and MTs play crucial roles in maintaining whole-cell mechanical properties of SMCs, and while AFs act as an internal tension generator, MTs act as a tension reducer, and these contribute to intracellular force balance three-dimensionally.