Calcein was used in vitreous fluorophotometry (VF) to study the blood-retinal barrier (BRB) in rats with streptozocin-induced diabetes. Calcein was injected intravenously in 18 diabetic rats and sodium fluorescein (NaF) in 12 diabetic rats. VF was performed at 1, 2, 4, 6, 9, and 21 hours following the single injection in these diabetic rats; it was also performed at these times in 24 normal (control) rats. On another day, the same experiment was performed using NaF. There was no significant difference between the two dyes in the diabetic and nondiabetic rats at 1 hour. However, at 4 hours and at later measurement points, there was dye leakage into the vitreous, peaking at 9 hours; the leakage was significantly greater in the diabetic rats. Also, the leakage was significantly greater with calcein than it was with NaF. The data demonstrate that calcein circulates longer and at higher levels than NaF and thus is probably more useful for BRB integrity studies.