The concentrations of furosemide (FU) in sequential samples from scala tympani (ST) and scala vestibuli (SV) perilymph after intravenous injection of 100 mg/kg FU were measured by high-performance liquid chromatography (HPLC) with or without blocking the cochlear aqueduct (CA). An analysis of amino acid content in perilymph was also perfomred by HPLC using phenyl isothiocyanate derivatization as the sample volume withdrawn through the round window (RW) or the hole in the third turn of the cochlea was varied from 100 nl to 5 μl. We found pronounced gradient and difference of FU concentrations between ST and SV perilymph. The elimination curve of FU in ST, however, became very similar to that in CSF as the CA was not blocked. Up to 200 nl of sample volume, the glycine and alanine contents of ST perilymph was similar to that of SV perilymph. With larger volumes, the glycine and alanine contents rapidly declined toward that of CSF. Our data suggest that perilymph samples taken through the RW without blocking the CA are contaminated with CSF if more than 200 nl is withdrawn. If ST perilymph is sampled from the third turn even without blocking CA, larger volumes (up to 1.7 μl) can be withdrawn without significant CSF contamination.