The objective of this study was to establish a nonradioactive technique to accurately assess blood flow in a small tissue such as the anterior optic nerve. Colored microspheres, 10.2 +/- 0.23 microns in diameter, were injected into the left atrium in each of 17 anesthetized rabbits. The rabbits were divided into four groups injected, respectively, with 5, 10, 50, or 100 million microspheres. Microsphere quantification in the tissue was performed after postmortem dissection and alkaline corrosion of the anterior optic nerve. Blood flow was evaluated by means of reference sample comparison method. While the relative interocular difference (range, 15.3-162.8%) was not significantly different between the rabbits injected with 5, 10, or 50 million microspheres (Kruskal-Wallis test, P = 0.11), injection with 100 million microspheres yielded a significantly lower relative interocular differences (range 2.3-12.8%) compared to the other three groups (Kruskal-Wallis test, P = 0.0048). In addition, reproducibility of microsphere counts was evaluated by ranking the difference between right and left optic nerve in percentage of the average number of microspheres per milligram of tissue in both optic nerves. The correlation between the relative interocular difference (range, 2.3-162.8%) and the average number of microspheres per milligram of tissue (1.2-78.7 microspheres/mg optic nerve tissue) was statistically significant (Spearman R, -0.90; P < 0.0001). The interocular variability in microsphere counts and interocular difference in intraocular pressure did not correlate (Spearman R, 0.33; P = 0.20). The rabbits injected with 100 million microspheres showed the highest average number of microspheres (range, 31.8-78.7 microspheres/mg optic nerve tissue). The optic nerve blood flow ranged between 0.14 and 0.24 microliters/mg/min among the rabbits injected with 100 million microspheres. The present experimental technique of optic nerve blood flow measurement is relatively inexpensive, highly reproducible, and obviates disposal of radioactive materials.