Choroid plexus epithelial cells react to changes in osmolarity by passive cell volume changes as other mammalian cells. In previous studies, hyperosmolar challenges resulted in cell shrinkage when either mannitol or NaCl was applied to increase osmolarity. However, a robust cell swelling, despite an outward osmotic gradient, was reported upon challenge with KCl-induced hypertonicity. This was interpreted as inward obligate water transport with Na+, K+, and Cl− mediated by NKCC1. In the present study, single cell volume changes were recorded by pinhole calcein-fluorescence microscopy and contrast imaging before and after 1-minute challenges with osmolarity change. Mannitol- and NaCl-induced hyperosmolarity of 30 mOsm induced the expected ~10% reductions in cell volume by both methods (n=9). Similar KCl-induced hyperosmolarity led to three different responses: cell shrinkage by ~6%, cell swelling by ~50%, or a combined pattern with shrinkage followed by swelling (n=9). Some of the swollen cells started blebbing and/or disrupted, while others partially regained cell volume even after blebbing. KCl-induced cell swelling was sensitive to the NKCC1 inhibitor bumetanide (10 μM), indicating the dependence of the swelling on inward transport by this mechanism. The observation that cell swelling can occur despite a preceding shrinkage indicated that the water influx did not occur against an osmotic gradient. Another observation against NKCC1-mediated water transport was a robust ~14% cell shrinkage induced by 100 mOsm NaCl hyperosmolarity (n=5). Under these conditions, the inward chemical gradient for ions transported by NKCC1 are of similar magnitude as under 15 mOsm KCl-induced hyperosmolarity, where a subset of cells reacted with ~12% swelling (the majority of cells were shrinking, n=5). Thus, the cell swelling is not driven by the combined gradients for Na+, K+, and Cl− into the cells, but is probably secondary to a KCl-induced cell depolarization. Interestingly, cell depolarization by the monovalent cation ionophore gramicidin (10 μM) induces a Na+-dependent cell swelling in a subset of cells with similar magnitude and duration as KCl-induced hyperosmolarity (n=5). Thus, the heterogeneous response to KCl-induced hyperosmolarity may rely on variations in the initial membrane potential among isolated cells. In conclusion, this study can reproduce many of the previous observations regarding cell volume responses to hyperosmolarity in choroid plexus epithelial cells including a bumetanide-sensitive swelling phenomenon in a subset of cells. However, the ionic gradients are not driving this cell swelling, but is rather a consequence of cell depolarization. The research was supported by the Independent Research Fund Denmark | Medical Sciences and Aarhus University Research Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.