Using the tandem scanning microscope, in vivo confocal microscopic images of living eyes were compared to images obtained from ex vivo, freshly enucleated or fixed tissue in the rabbit. In the normal cornea, microscopic details of the superficial epithelium, basal lamina, stromal fibrocyte nuclei, nerves and endothelial cell borders were easily discernible. Removal of the eye from the intact animal resulted in loss of detail with distortion of the normal structural interrelationships within the corneal stroma whilst enhancing details of the corneal epithelium. Formalin fixation further enhanced details of the basal and suprabasal corneal epithelial cell nuclei and the stromal fibrocyte cell borders whilst inducing prominent brightly reflecting folds in the thickened stroma with concomitant enhancement of the edge contrast of the collagen lamellae. These changes appeared to be related, in part, to hydration of the cornea and artefactual pooling of water between structures that may enhance reflectivity by increasing the difference between the refractive index of the cellular and extracellular elements. We conclude that microscopic examination of ex vivo preparations of corneal tissue, although providing increased resolution similar to conventional light microscopic techniques, significantly altered the normal structural relationships and could lead to erroneous measurements of the physiological properties of the tissue as compared to in vivo microscopy of undisturbed, intact tissue.