A relatively simple method for the determination of the diffusion coefficient of a substance that has been injected into tissue is described. We illustrate this method using [ H-3] dexamethasone injected into the subcutaneous tissue of rats. Digital autoradiography was used to measure the distribution of the [ H-3] dexamethasone within the subcutaneous tissue at 2.5 and 20 min after injection. Measured concentration profiles of the injection were compared to a mathematical model of drug diffusion from an injection. There was good agreement between the experimental data and the mathematical model. The diffusion coefficient found using this simple injection method was ( 4.01 +/-2.01) x 10(-10) m(2)/s. This D value was very close to the value of D = ( 4.11 +/- 1.77) x 10(-10) m(2)/s found previously using different mathematical and experimental techniques with osmotic pumps implanted for 6, 24, and 60 h in rats ( 1). The simple method given here for the determination of the diffusion coefficient is general enough to be applied to other substances and tissues as well.