Rats are increasingly used in ophthalmic research. However, little is known about the metabolic regulation of ocular blood flow. The purpose of this study was to examine the vasoreactivity in retina and choroid of the rat eye in response to experimentally altered partial arterial pressure of CO2 (PaCO2). The retinal and choroidal blood flows were measured sequentially in different PaCO2 with a modified microspheres method. The experiments were performed in two groups of adult male Brown-Norway rats. Under isofluorane anesthesia and mechanical ventilation, PaCO2 was monitored continuously by recording end tidal carbon dioxide level. Both femoral arteries and a femoral vein were cannulated for arterial blood pressure monitoring, blood sampling and drug administration, respectively. The intraocular pressure in both eyes was manometrically controlled at 20 mmHg by anterior chamber cannulation. The retinal and choroidal blood flows were simultaneously measured by cardiac injection of a mixture containing 3.75 million of 8 μm, and 0.5 million of 10 μm microspheres; each size having a distinct color. In one experiment (n = 10), blood flow was first measured during normocapnia (PaCO2 = 35 mmHg) and then during hypocapnia (PaCO2 = 20–25 mmHg). In another experiment (n = 7), blood flow was measured during hypercapnia (PaCO2 = 45–50 mmHg) and repeated one more time under the same experimental conditions to evaluate the repeatability of sequential measurements and the variances of the measurement between the two eyes. The results show that the mean blood flow in the retina measured during hypocapnia, normocapnia and hypercapnia were 8.1 ± 4.8, 15.1 ± 8.5 and 27.4 ± 4.6 μl/min per tissue, respectively. In the choroid, the corresponding blood flow rates were 120 ± 38, 166 ± 28 and 149 ± 28 μl/min per tissue, respectively. The difference of the mean blood flows across all the three different PaCO2 groups was highly significant for both retina and choroid (ANOVA: P < 0.0001 and P = 0.01, respectively). The mean blood flow during hypocapnia was significantly lower than normocapnia in both retina and choroid (P < 0.02). The blood flow under hypercapnia was significantly higher than normocapnia in retina (P < 0.01), but not in choroid (P = 0.62). In conclusion, the study demonstrated that the dual-size and dual-dose microspheres mixture can be used as a reliable method to measure the retinal and choroidal blood flows simultaneously and sequentially in rats. The vasoreactivity to altered systemic PaCO2 in the retina in rats is similar to that of most other species studied. However, the choroidal vascular system exhibited complicated features that remain to be further clarified.