Cell-based drug delivery systems have generated an increasing interest in recent years. We previously demonstrated that systemically administered macrophages deliver therapeutics to CNS, including glial cell line-derived neurotrophic factor (GDNF), and produce potent effects in Parkinson's disease (PD) mouse models. Herein, we report fundamental changes in biodistribution and brain bioavailability of macrophage-based formulations upon different routes of administration: intravenous, intraperitoneal, or intrathecal injections. The brain accumulation of adoptively transferred macrophages was evaluated by various imaging methods in transgenic Parkin Q311(X)A mice and compared with those in healthy wild type littermates. Neuroinflammation manifested in PD mice warranted targeting macrophages to the brain for each route of administration. The maximum amount of cell-carriers in the brain, up to 8.1% ID/g, was recorded followed a single intrathecal injection. GDNF-transfected macrophages administered through intrathecal route provided significant increases of GDNF levels in different brain sub-regions, including midbrain, cerebellum, frontal cortex, and pons. No significant offsite toxicity of the cell-based formulations in mouse brain and peripheral organs was observed. Overall, intrathecal injection appeared to be the optimal administration route for genetically modified macrophages, which accomplished targeted gene delivery, and significant expression of reporter and therapeutic genes in the brain.