Kinetics of hydrophilic solute entry into endolymph (EL), perilymph (PL), and cerebrospinal fluid (CSF) were studied after intravenous administration (sodium, urea, glycerol, mannitol, sucrose) and cerebral lateral ventricle injection (urea, sucrose) of tracers in anesthetized rats. Samples of cochlear EL, PL of scala vestibuli (PLV), PL of scala tympani (PLT), and cisternal CSF were obtained. The data showed slow entry of tracers in PLV, PLT, and CSF as follows: Na greater than urea greater than mannitol approximately sucrose; slower entry of mannitol and sucrose in PLT and CSF than in PLV; 1 h delayed peak of radioactivity in PLV compared with the immediate peaks in PLT and CSF after CSF injection, and the value of PLV peak was 13% that in CSF; extremely slow entry of nonelectrolytes in EL. These results indicate that PLV originates mainly from plasma across a blood-perilymph barrier that restricts the entry of small hydrophilic solutes. The blood-perilymph barrier is most likely composed of an endothelial barrier associated with an epithelial secretion. The latter could be located at the vasculo-epithelial zone of the spiral limbus.