Smooth muscle cells derived from rat aortic media were established in subcultures and seeded from suspensions on smooth silicone rubber substrata in tissue culture flasks. Each flask contained two substrata, one stationary and one that was stretched and allowed to recoil at 15-s intervals. After 48 h or longer, the cells were fixed and their pattern on the 2 substrata examined by light or scanning electron microscopy. All stretched substrata showed 75% or more of cells lying within 45° of a line drawn at right angles to the direction of stretch and recoil. Cells on the stationary substrata showed a random orientation. The results suggest that stretching of the internal elastic lamina by arterial pulsation may be a factor in creating the characteristic longitudinal orientation of smooth muscle cells that appear in the subendothelial space after injury, diffuse intimal thickening or atherosclerosis.