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This study modified the microspheres method by optimizing the dose and size of microspheres (MS) to enable accurate ocular blood flow measurement in rats. Fluorescent MS, either 6, 8, 10 or 15 μm diameter, were administered into the left ventricle of anesthetized adult Brown Norway rat in a dose of either 106, 5 × 106, or 107. The total number of MS entrapped in retina, choroid and optic nerve (Ntissue) was quantified and compared between size and dose groups. The MS distribution in the retina and their reentry into systemic circulation were evaluated for different sized MS. The results showed that at the 5 × 106 dose, the Ntissue of 8 μm MS was significantly more than either 6 or 10 μm MS in the retina (P < 0.02) and optic nerve (P < 0.03). The 10 μm MS produced the highest Ntissue for the choroid, as compared with either 8 or 6 μm MS (P < 0.03). At the 106 dose, no difference of Ntissue was found between 8, 10, and 15 μm MS in the retina. The 10 μm MS yielded the highest Ntissue in the choroid as compared to 8 and 15 μm MS (P < 0.003). The Ntissue for 8 μm MS was higher than both 10 and 15 μm (P < 0.01) MS in the optic nerve. No MS (≥8 μm) reentered the systemic circulation. The 15 μm MS tended to lodge in pre-capillary arterioles and caused significant blood pressure increase during the injection. The blood flow measured with the optimal size MS (mean ± SE) were 19 ± 3.4 and 170 ± 35 μl/min in the retina and choroid, respectively; and 0.18 ± 0.03 μl/min per mm optic nerve. It is concluded that the 8 μm MS are the optimal size for both retinal and optic nerve blood flow estimation; the 10 μm for the choroid. The optimal dose for the retina was approximately 2.5 × 106, 0.5 × 106 for the choroid, and 5 × 106 ∼ 107 for the optic nerve. The 15 μm MS are inappropriate for ocular blood flow measurements in rats.