Corneal collagen ultrastructural changes occur during the healing process. The present study was designed to compare collagen ultrastructural changes after trephine wounding or flap creation. Bovine corneas were injured and maintained in organ culture for up to 4 weeks. Samples were removed from culture at 0, 1, 2, 3 and 4 weeks and snap frozen in liquid N2. X-ray scattering was used to measure changes in collagen interfibrillar spacing, intermolecular spacing and fibrillar diameter. Some samples were fixed in 10% Neutral Buffered Formalin solution and wax embedded for immunohistochemistry to monitor myofibroblast differentiation in corneal flaps. Swelling effects (i.e. changes in interfibrillar spacing) were more severe in trephined corneas than in those with stromal flaps. Collagen fibrillar diameter remained normal in the periphery of injured corneas, but increased significantly in areas within and around the wound in trephined samples and in the epithelial incision site in corneal flaps. Intermolecular spacing was unchanged in all samples. In the flaps, αSMA expression was only detected in an area adjacent to the epithelial plug, and cell numbers gradually increased during the culture. We conclude that stromal swelling is more rapid for trephine-wounded corneas than in stromal flaps, indicating that the intensity of the corneal healing response depends on the type of injury. © 2008 Elsevier Ltd. All rights reserved.