We evaluated alkaline-solubilized collagen, hyaluronic acid (HA), and a substance formed from mixing both materials as vitreous substitutes in the rabbit. Fluorescein isothiocyanate (FITC)-labeled collagen (2%), fluorescein-labeled HA (FL-HA, 1%), or the mixture was injected into the rabbit vitreous after vitrectomy. After 3 months, histologic study revealed that the substitutes caused no adverse effects on the ocular tissue. Kinetic studies showed that the half-lives of FITC-collagen, FL-HA, and the mixed substance were 5.70, 2.09, and 8.41 days, respectively. The results indicated that HA enhances the collagen half-life. The concomitant of collagen and HA is safe and effective for 3 months in rabbit’s eye as a vitreous substitute, and the mixture is capable of tamponade.