Glial cell line-derived neurotrophic factor (GDNF) is produced both in the central nervous system (CNS) and the periphery. Effective in ameliorating neurodegeneration in several animal models of CNS disease, its promise as a therapeutic agent would be greatly enhanced if it readily crossed the blood-brain barrier (BBB) in unmodified form. Here, we used the sensitive techniques of multiple-time regression analysis and ex-vivo perfusion in blood-free buffer to examine the entry of (125)I-GDNF into mouse brain. The integrity of GDNF in blood and brain was examined by high performance liquid chromatography and the physicochemical properties determining permeability were measured by octanol/buffer partition coefficient and hydrogen bonding. The efflux of (125)I-GDNF was determined to test for the presence of a bidirectional transport system. The results show that (125)I-GDNF differs from other peptides and polypeptides in that it does not enter brain any faster than (99m)Tc-albumin, an effect that cannot be explained by degradation, rapid efflux, protein binding, or inadequate lipophilicity. Thus, GDNF shows a different type of interaction with the BBB. In normal mice, the BBB functions as a substantial physical barrier; in pathological or traumatic situations when the barrier is partially disrupted, the lack of restriction by a saturable transport system could make GDNF a suitable candidate for peripheral delivery in promoting neuroregeneration.