PURPOSE. An in situ forming hydrogel was evaluated as a potential vitreous substitute in rabbits. METHODS. The hydrogel used a disulfide cross-linker that was then reduced to produce an injectable thiol-containing polymer solution. The disulfide cross-links reformed by air oxidation of the thiols and produced a stable hydrogel once inside the eye. The polymer was clear, autoclavable, and could be stored easily in the presence of nitrogen gas. Capillary rheometry was used to measure the viscoelastic properties of the hydrogels and the porcine vitreous. Fourteen black rabbits underwent a pars plana, 25-gauge, three-port vitrectomy by a single surgeon with injection of a vitreous substitute. RESULTS. The refractive indices of the hydrogels were measured by refractometry and were shown to be close to 1.33, and the 2% hydrogel matched the mechanical properties of the natural vitreous humor. The reduced polymeric hydrogel was easily injectable through a small-gauge needle into the vitreous cavity and did not show any fragmentation. The material underwent gelation within the eye, remained optically clear, and appeared well tolerated clinically. Slit lamp examination, dilated fundus examination, and electroretinograms showed no evidence of vitritis, uveitis, or endophthalmitis after 1 week. Histopathologic evaluation did not reveal any overt toxicity or gross morphologic changes in the retina. CONCLUSIONS. The fact that this process of in situ gelation gives rise to hydrogels that are biocompatible and physically and optically similar to the natural vitreous suggests its suitability as a permanent vitreous substitute. Hydrogel candidates will be further studied to evaluate long-term biocompatibility and degradation in vivo.