We tested the hypothesis that, during acute glucose-induced hyperosmolality, the brain shrinks less than predicted based on an ideal osmometer, and volume regulation is present in fetuses, premature and newborn lambs. Brain water responses to glucose-induced hyperosmolality were measured in the cerebral cortex, cerebellum and medulla of fetuses at 60% of gestation, premature ventilated lambs at 90% of gestation, newborn lambs and adult sheep. After exposure of the sheep to increases in osmolality with glucose plus NaCl, brain water and electrolytes were measured. The ideal osmometer is a system in which impermeable solutes do not enter or leave in response to an osmotic stress. In the absence of volume regulation, brain solute remains constant as osmolality changes. The osmotically active solute demonstrated direct linear correlations with plasma osmolality in the cerebral cortex of the fetuses at 60% of gestation (r=0.72, n=24, P=0.0001), premature lambs (r=0.58, n=22, P=0.005), newborn lambs (r=0.57, n=24, P=0.004), and adult sheep (r=0.70, n=18, P=0.001). Similar findings were observed in the cerebellum and medulla. Increases in the quanity of osmotically active solute over the range of plasma osmolalities indicate that volume regulation was present in the brain regions of the fetuses, premature lambs, newborn lambs and adult sheep during glucose-induced hyperosmolality. We conclude that, during glucose-induced hyperosmolality, the brain shrinks less than predicted based on an ideal osmometer and exhibits volume regulation in fetuses at 60% of gestation, premature lambs, newborn lambs and adult sheep.