Abstract A highly sensitive, reproducible, and rapid gas chromatographic method for ethanol determination in various biological specimens (human whole blood, serum, urine, and fecal supernatants) was developed. The method involves direct injection of the biological specimen into the gas chromatograph, without any pretreatment. Contamination of the gas chromatographic column with nonvolatile material was prevented by the use of a glass liner in the injector. This liner, which acted as a precolumn, was partly filled with small glass beads. Injection was performed in between the glass beads. More than 50 injections of the various biological specimens could be done before the liner had to be replaced by a new one. This injection technique between glass beads allows direct injection of large sample volumes up to 10 μL without disturbing the gas chromatographic separation. Injection of these large sample volumes made the method very sensitive. The detection limit for ethanol amounted to 0.1 mg/L (2 μmol/L) when using an injection volume of 5 μL. Attention has also been paid to simultaneously monitoring ethanol, methanol, acetaldehyde, and acetone in blood and urine of control subjects.