Background <p>Cerebrospinal fluid (CSF) is vital to maintain brain homeostasis by facilitating waste removal, nutrient transport, and regulation of intracranial pressure. The choroid plexus is generally assigned as the primary source of CSF secretion, but it remains unresolved to what extent water produced during cellular metabolism in the central nervous system contributes to the CSF pool.</p> Methods <p>Brain and spinal cord parenchyma volumes in rats (<i>n</i> = 6) and humans (<i>n</i> = 6) were assessed with volumetric magnetic resonance imaging. Segmentation techniques were applied to obtain the mass of metabolically active tissue. The volume of H₂O produced as a biproduct of glucose oxidation was estimated based on the established oxygen consumption of these tissues.</p> Results <p>We estimate a metabolic H₂O production within the brain and spinal cord tissue of 0.078 µL/min in rats and 28.5 µL/min in humans, which amounts to 1–8% of the volume of CSF secreted into the ventricular compartment.</p> Conclusions <p>Our results demonstrate a minor contribution of the metabolically produced water to the collective CSF pool in rats and humans, notwithstanding that part of the estimated H₂O production is likely absorbed by other H₂O-utilizing enzymatic processes. Our findings thus support the longstanding view that the choroid plexus is a key contributor to CSF secretion, with potential implications for future understanding and management of pressure-related brain disorders.</p>