In alloxan diabetic rabbits, plasma osmolality was acutely elevated from about 309 to 365 mOsm/kg by intravenous infusion of either glucose or mannitol, and effects upon brain and cerebrospinal fluid (CSF) water and solute were evaluated. After 4 hr of hyperglycemia, plasma osmolality was then rapidly (3 hr) lowered to control values with either insulin or glucose free peritoneal dialysis. It was found that during 4 hr of hyperglycemia, the brain initially (2 hr) lost significant quantities of water (8.4%) but after 4 hr, brain water content had returned to normal and osmolalities of plasma, CSF and brain were similar. The increase in brain osmolality during hyperglycemia was only partially due to increases in concentration of measured solutes (Na+, K+, C1−, glucose, lactate, sorbitol, amino acids); most of the increase (72%) was due to the presence in brain of undetermined solutes (idiogenic osmoles) which were not glucose metabolites. During mannitol infusion, however, essentially all of the increase in brain osmolality was due to a loss of brain water. When plasma glucose was rapidly lowered from 78 to 28 mm with insulin, an osmotic gradient developed between brain (339 mOsm/kg H2O) and plasma (307 mOsm/kg). Further lowering of plasma glucose below 14 mm resulted in a significant increase in brain K+ (13%) and a net movement of water into brain, causing cerebral edema. Lowering plasma osmolality with peritoneal dialysis did not increase brain K+ or cause brain edema. It is concluded that when plasma osmolality is elevated by mannitol infusion, brain osmolality is increased secondary to loss of brain water. When plasma osmolality is elevated by hyperglycemia, however, brain osmolality increases largely by generation of idiogenic osmoles, without significant change in brain water or electrolyte content. When plasma glucose in hyperglycemic animals is rapidly lowered with insulin, an osmotic gradient develops between brain and plasma, but cerebral edema does not occur unless plasma glucose is lowered to near normal levels.