Unilateral rhegmatogenous retinal detachments were created in seven cynomolgus monkeys. Six months later, aqueous, vitreous, and subretinal fluid protein concentrations were measured. In fellow eyes with vitrectomy alone, mean aqueous and vitreous protein concentrations were 0.28 and 0.23 mg/mL, respectively. In eyes with retinal detachments, mean aqueous, vitreous, and subretinal protein levels were 1.46, 2.66, and 4.74 mg/mL, respectively. Eyes with a large retinal hole (greater than or equal to 1 disc diameter) had a subretinal fluid-vitreous protein concentration ratio of 1.1, indicating free diffusional exchange between the vitreous and subretinal space. In eyes with a small retinal hole (less than or equal to 1/4 disc diameter), the corresponding ratio was 4.0. These findings are consistent with the hypothesis that fluid moves from the vitreous cavity through the retinal hole into the subretinal space, preventing back diffusion of protein from the subretinal space into the vitreous, and allowing accumulation of protein in the subretinal space.