We compared quantitative experimental results on the diffusion of 35S-labeled phosphorothioate oligonucleotide (PS-ODN) after intraparenchymal infusion in rat brain, with the distributions predicted by Fick's second law of diffusion. Fischer 344 rats underwent identical intracerebral infusions of 36S-PS-ODN. After 0, 5, 11, 23, and 47 h, groups of animals were sacrificed and sequential brain cryosections subjected to autoradiography. The resulting experimental data were compared to the predicted distributions, for estimation of the apparent free diffusion coefficient, D*. Volumes of distribution and total content of 36 S-PS-ODN in the parenchyma were also computed, to monitor loss of total material. The values for D* were within the expected range for the 21-mer PS-ODN used, but a progressive decrease in D* over time was noted. The model requires D* to remain constant and, thus, does not adequately explain the spread of 35S-PS-ODN following infusion. The progressive slowing of spread over time suggests that at later time points, 35S-PS-ODN may be fixed by tissue binding or cellular uptake in the brain. Loss of material via vascular and CSF clearance may also contribute to the lack of fit. Our results highlight issues to be addressed in the modeling and experimental design of the intraparenchymal infusion process.