Astrocytes are, as normal constituents of the brain, promising vehicles for ex vivo gene delivery to the central nervous system. In the present study, we have used a lentiviral vector encoding glial cell line-derived neurotrophic factor (GDNF) to transduce rat-derived primary astrocytes, in order to evaluate their potential for long-term transgene expression in vivo and neuroprotection in a rat model of Parkinson's disease. Following transplantation of GDNF-transduced astrocytes to the intact striatum, the level of released GDNF was 2.93 ± 0.28 ng/mg tissue at 1 week post-grafting, reduced to 0.42 ± 0.12 ng/mg tissue at 4 weeks, and thereafter was maintained at this level throughout the experiment (12 weeks; 0.53 ± 0.068 ng/mg tissue). Similarly, grafting to the substantia nigra (SN) resulted in a significant overexpression of GDNF (∼0.20 ng/mg tissue) at 1 week. Intact animals receiving transplants of GDNF-transduced astrocytes displayed an increased contralateral turning (5.39 ± 1.19 turns/min) in the amphetamine-induced rotation test, which significantly correlated with the GDNF tissue levels measured in the striatum, indicating a stimulatory effect of GDNF on the dopaminergic function. Transplantation of GDNF-transduced astrocytes to the SN 1 week prior to an intrastriatal 6-hydroxydopamine lesion provided a significant protection of nigral tyrosine hydroxylase-positive cells. By contrast, when the cells were transplanted to the striatum, the level of released GDNF was not sufficient to rescue the striatal fibers and, hence, to protect the nigral dopaminergic neurons. Overall, our results suggest that genetically modified astrocytes expressing GDNF can provide neuroprotection in a rat model of Parkinson's disease following transplantation to the SN.