The aim of this work is to develop a technique to accurately measure the diffusion coefficient of drugs and drug surrogates in the vitreous humor using MRI. Fresh bovine eyes were used for drug diffusion study in the vitreous, and Gd-DTPA was chosen as a drug surrogate/contrast agent to visualize the diffusion process by MRI. Experiments were conducted by injecting 30 mu l of the Gd-DTPA with a concentration of 20 mM/l in 0.9% saline in the vitreous of the whole bovine eye. MRI images were acquired at regular intervals for 1.5-2 h, and Gd-DTPA concentration was determined from the MRI signal intensity. At each time point, concentration contours were constructed and a least-squares best fit to the corresponding theoretical contours, based on a cylindrical bolus model, was performed. The best fit at different time points resulted in fairly consistent diffusion coefficient values. With the surrogate injection technique perfected, highly-symmetric distribution of Gd-DTPA was observed, allowing for spherically symmetric mathematical models, with adjustments for ellipticity as needed, for the diffusion coefficient analysis. The analysis yielded an average diffusion coefficient value of (3.040 +/- 0.274) x 10(-6) cm(2)/s. The 3-D MRI visualization together with careful symmetric injection of the surrogate/contrast agent has provided a quantitative tool for the accurate measurement of the diffusion coefficient. With symmetric injection and corresponding diffusion theory based on the point-source model with adjustments for nonzero bolus size and asymmetry, strong agreement of theory with experiments has been achieved. (C) 2013 Elsevier Ltd. All rights reserved.