OBJECTIVE To evaluate changes in the donor corneal endothelium in the intact globe and in the in vivo rabbit cornea to characterize more fully the formation of "dark events" without relief images in the endothelial mosaic. METHODS Six ex vivo human donor corneas in the intact globe and an in vivo rabbit model were used to assess the morphological changes associated with osmotically increasing fluid movement from the anterior chamber into the stroma by specular (SM), confocal (CM), and transmission and scanning electron microscopy (TEM, SEM). RESULTS After application of hyperosmotic solution on the anterior surface of the cornea, dark events without relief images were observed by SM and CM. In both human and rabbit corneas, SEM showed that apical pores at the Y-junctions between endothelial cells became enlarged. Large subendothelial spaces were observed on Descemet's membrane by TEM with some spaces communicating with the anterior chamber. CONCLUSIONS The findings suggest that these openings at the Y-junctions may represent intercellular channels that may also act as pathways for the formation of intercellular and subendothelial vacuoles in both the rabbit and human donor corneal endothelium. By virtue of their location, these vacuoles are characterized by lack of relief images as seen with the contact SM.