The authors previously developed a new model of preretinal neovascularization in the rabbit eye using hyaluronidase for enzymatic vitreolysis. The purpose of this study was to evaluate the safety of intravitreal injections of hyaluronidase. Concentrations of 1, 15, 30, 50, and 150 IU of hyaluronidase in 0.1 ml of 0.9% saline were injected intravitreally and aspirated repetitively until the vitreous was partially liquified. The animals were examined with indirect ophthalmoscopy, fundus photography, and fluorescein angiography before injection and on days 1 and 7 after injection. Light and electron microscopic retinal sections were prepared from enucleated eyes at days 1 and 7. All concentrations of hyaluronidase were effective in producing partial vitreolysis. Eyes treated with 1 IU showed no abnormalities on days 1 or 7. Eyes treated with 15 IU showed no retinal abnormalities on day 1, but on day 7 histologic abnormalities were present in two of four eyes. At higher concentrations, clinical and histologic changes were seen in proportion to the concentration and included focal whitening, edema, vitreous haze, vascular abnormalities, and retinal necrosis at the highest doses. Histologic evaluation of the retina revealed marked destruction in all layers at the higher concentrations. The authors conclude that 1 IU of intravitreal hyaluronidase is sufficient for partial vitreolysis and nontoxic to the rabbit retina.