Cerebrospinal fluid (CSF) secretion into the brain ventricular system is an active process via the choroid plexus (CP) – a specialized, convoluted epithelial sheet surrounding a fenestrated capillary bed which is fed from the cerebral arterioles. The final CSF composition differs from a simple plasma filtrate implicating the CP epithelial cells (CPe) as an important regulatory site controlling CSF composition and volume. Solute and water transport across the blood-CSF barrier are tightly controlled by ion and water transport proteins. Transient Receptor Potential Vanilloid 4 (TRPV4) is a polymodal, non-selective cation channel which functions as a regulatory hub protein in epithelia, including the CP. Previous studies from our group demonstrated d that TRPV4-selective antagonists were effective at reducing ventriculomegaly in a genetic rat model of severe postnatal hydrocephalus, a condition resulting from excess CSF (Hochstetler and Smith et al., 2020). In CPe, it was found that activation of TRPV4 caused the influx of both Ca2+ and Na+ ions, and entry of both of these ions was selectively blocked by a TRPV4-selective antagonist. Systemic treatment with TRPV4 antagonists reduced TRPV4 mRNA expression in the CP in hydrocephalic rats, but not in normal rats, indicating a role for TRPV4 as part of a regulatory pathway in CSF production under pathological conditions. To study the mechanism of CSF secretion by the CP, we utilized two continuous, high resistance CP cell lines: the porcine choroid plexus – Reims (PCP-R) and the human choroid plexus papilloma (HIBCPP) cells. The current experiments utilize Ussing chamber electrophysiological techniques, western blotting, RT-PCR, and fluorescent immunolabeling. Immunolabeling and mRNA expression demonstrate a robust abundance for TRPV4 and other well-characterized membrane proteins in both cell lines. In the lines, TRPV4 activation stimulates a large change in short circuit current (ISC), a measure of net electrogenic ion flux. This change in ISC is coupled with a substantial change in transepithelial membrane conductance, a measure of cellular permeability. The transepithelial membrane transport changes due to TRPV4 activation for human and porcine cells are similar in overall effect, but differ in baseline, duration, and magnitude, indicating the possibility of species-specific CP transport mechanisms. TRPV4 activation can be inhibited by pre- incubation with TRPV4 antagonists, and the changes in ion flux and conductance can be reversed by addition of TRPV4 antagonists after initiation of the response, these studies substantiate the specificity of the mediator-induced changes and provide an in vitro model for testing potential drug actions. Furthermore, in both the porcine and human cells, the TRPV4 response is dependent upon K+ secretion, Cl- absorption, and Na+ secretion. These data confirm a role for TRPV4 in regulating ionic gradients, thereby modulating both electrogenic and electroneutral transporters, and controlling CSF production. The promising results of these studies, coupled with published data from other investigators suggesting a role for TRPV4 in blood-brain-barrier permeability and astrocyte volume regulation, shows the importance of TRPV4 as a regulatory hub protein in maintaining brain fluid volume.