A rapid and highly specific method for determination of serum estetrol during pregnancy and at delivery is developed using a gas chromatography-mass spectrometry with application of newly synthesized deuterated estetrol as an internal standard. Evaluation of the method assessed by recovery experiments was 102.4 and 103.0 percent when 2 and 4ng of estetrol was added to 0.5ml of male serum. The coefficient of variation were 2.54 and 2.84 percent, respectively. The results obtained by the present method are correlated well with those obtained by conventional RIA. Serum unconjugated estetrol levels during pregnancy increased with progressing gestation. The levels from the 36th to the 40th week were 749.8 +/- 281.5pg/ml (mean +/- S.D.) The levels in umbilical cord blood at delivery were also measured: Umbilical vein were 8,064.0 +/- 6,595.4pg/ml which were statistically higher than in umbilical artery (3,515.9 +/- 1,553.8pg/ml) and in maternal peripheral vein (1,209.0 +/- 530.3pg/ml).