Choroid plexus epithelial cells form an integral and important part of the barrier between blood and cerebrospinal fluid. Culture of choroid plexus epithelium in vitro has been achieved from several mammalian species and this provides opportunities for the study of choroid plexus development and function, including the capacity of the epithelial cells to control the movement of bioactive molecules, such as novel drug candidates, from the bloodstream to the brain. Here we describe a method for the derivation of primary cell cultures from mouse choroid plexus epithelium, together with characterisation by immunofluorescence using antibodies specific to markers of mature choroid plexus epithelial cells. With this method, relatively pure choroid plexus epithelial cell monolayers are established using the DNA synthesis inhibitor cytosine arabinoside (Ara-C), which is cytotoxic to contaminating cell types such as fibroblasts, but not the epithelial cells. These cells are shown to express the diagnostic choroidal marker, transthyretin (TTR), as well as markers of epithelial cell differentiation and are thus suitable for studies that address the transport and barrier functions of the choroid plexus.