The inorganic phosphate (P_i) concentration of mammalian cerebrospinal fluid (CSF) is about one-half that of plasma, a phenomenon also shown here in the spiny dogfish, Squalus acanthias. The objective of the present study was to characterize the possible role of the choroid plexus (CP) in determining CSF [P_i]. The large sheet-like IVth CP of the shark was mounted in Ussing chambers where unidirectional ³³P_i fluxes revealed potent active transport from CSF to blood side under short-circuited conditions. The flux ratio was 8:1 with an average transepithelial resistance of 87 ± 17.9 x cm² and electrical potential difference of +0.9 ± 0.17 mV, CSF side positive. The active P_i absorption from CSF was inhibited by 10 mM arsenate, 0.2 mM ouabain, Na⁺-free medium, and by increasing [K⁺] from 5 mM to 100 mM. Li⁺ stimulated transport 2-fold compared to Na⁺-free medium. Phosphonoformic acid (1 mM) had no effect on active P_i transport. RT-PCR revealed both PiT1 and PiT2 (SLC20 family) gene expression, but no NaPiII (SLC34 family) expression, in the shark CP. PiT2 immunoreactivity was shown by immunoblot, and localized by immunohistochemistry in (or near) the CP apical microvillar membranes of both shark and rat. PiT1 appeared to be localized primarily to the vascular endothelial cells. Together, the data indicate that the CP actively removes P_i from the CSF. This process has transport properties consistent with a PiT-2, Na⁺-dependent transporter which is located in the apical region of the CP epithelium.