Using an automated HPLC system reported recently by us (J Clin Lab Anal 4:410-413, 1990), serum unconjugated estriol and estradiol (u-E3 and u-E2) can be measured within 22 min. The clinical value of this method was evaluated in this study with abnormal pregnancies, twin pregnancies, and pregnancies associated with fetal anencephaly or complicated by gestational toxicosis or diabetes mellitus. A significant correlation was demonstrated between an abrupt decrease in serum u-E3 level and an abnormal pattern on fetal heart rate monitor. Since urinary E3 was not sensitive enough to detect any depressed fetal condition, serum u-E3 is better biochemical parameter for assessment of fetal well-being. In addition, u-E2 was measured in the sera of nonpregnant women with separation chromatography as a pretreatment in order to obtain higher sensitivity of the system. This modified HPLC system enabled us to determine the serum u-E2 in about 60 min with the minimal detectable value of 80 pg/ml; the coefficient of correlation with RIA was 0.822. As a result, this quick measurement of u-E3 and u-E2 by our HPLC system revealed to be useful not only for evaluating fetoplacental function but also for monitoring ovarian follicle maturation.