Introduction: The arachnoid tissue is a critical component of cerebrospinal fluid removal. Failure of that function results in hydrocephalus, a serious medical condition. The purpose of this study was to characterize arachnoid cell transport in culture and on three-dimensional collagen scaffold. Methods: Arachnoid cells were harvested from rat brainstems and cultured onto bilayered bovine collagen scaffolds. Cell growth and phenotype (protein expression and morphometry) were determined. Permeability and hydraulic conductivity were quantified. Results: Cells harvested from the anterior brainstem surface exhibited arachnoid cell phenotype (positive for vimentin, desmoplakin, and cytokeratin), readily penetrated the collagen scaffold, and doubled approximately every 2-3 days. The transepithelial electrical resistance value for a monolayer of cells was 160 Ω cm2 and the permeability of indigo carmine was 6.7×10-6±1.1×10-6 cm/s. Hydraulic conductivity of the collagen construct was 6.39 mL/min/mmHg/cm2. Conclusion: Cells isolated from the anterior brain stem exhibited the same phenotype as those found in the native tissue and exhibited aspects of barrier function found in vivo. These studies suggest that an ex vivo model for the arachnoid granulation can be developed.