Diethylstilbestrol (DES) is readily extracted into toluene from biological fluids or tissue homogenates. Sodium carbonate is added to the initial mixture to eliminate potentially interfering substances. The toluene is extracted with a very small volume of aqueous trimethylphenylammonium hydroxide. This solution generates dimethyl DES in the vaporizer of a gas-liquid chromatograph. An internal standard, dienestrol (DI), is added at the beginning of the procedure and is partitioned and methylated in the same manner as DES. The DES and DI derivatives are well separated in less than 6 min on an ov-17 column. The entire analysis requires less than 15 min for a fluid specimen and less than 25 min for a solid tissue specimen. Seven samples can be analyzed each hour on a single column with a flame ionization detector. The relative standard deviations at levels from 2.5 to 100 ppm in bile are less than 5%. The lower limit of sensitivity is 8 ppb in a 1 ml bile sample.