Glycerol concentrations in CSF, serum and perilymph after intravenous administration (3.0g/kg) were measured by fluorometric direct assay. Less than 2μl of perilymph (scala tympani) sample was taken from the third turn of the guinea pig cochlea after blocking the cochlear aqueduct to prevent the CSF contamination. The concentratoins of glycerol in CSF and perilymph lagged behind the serum increase. The concentration of glycerol in CSF reached the maximum value within 30 minutes after administration. A peak concentration was obtained in perilymph at 60 minutes with a subsequent slow decline. The concentrations in perilymph were lower than those in CSF at any time after injection of glycerol. These results suggest the hydrostatic pressure in perilymph is reduced more effectively than in CSF through the injection of an osmotic agent (glycerol) into systemic circulation.