Maternal and umbillical arterial and venous plasma and amniotic fluid testosterone (T) and dihydrotestosterone (DHT) were measured by radioimmunoassay. Maternal plasma T was 690 +/- 80 pg. per milliliter (mean +/- S.E.) in early pregnancy (less than 20 weeks) and increased significantly (p = 0.0002) to 1,095 +/- 177 pg. per milliliter in late pregnancy (greater than 20 weeks). DHT was 113.0 +/- 18.8 pg. per milliliter in early pregnancy and 179.8 +/- 30.5 pg. per milliliter in late pregnancy. Both umbilical arterial (UA) and umbilical venous (UV) plasma T were significantly higher in 11 male infants (UA T = 135.6 +/- 16.5 pg. per milliliter; UV T = 227.5 +/- 40.8 pg. per milliliter) than in 12 female infants (UA T = 92.1 +/- 9.7 pg. per milliliter; UV T = 89.6 +/- 12.6 pg. per milliliter) (p = less than 0.05 and less than 0.005, respectively). UV DHT and UA DHT showed no significant difference between male and female neonates. In midtrimester pregnancy, amniotic fluid T (AFT) was 165.2 +/- 15.4 pg. per millitier in pregnancies with a male fetus and was significantly higher (p = less than 0.001) than in pregnancies with a female fetus (mean +/- S.E. = 27.6 +/- 2.6 pg. per millitier). In late pregnancy, AFT levels were similar to those of early pregnancy, but a considerable overlap in AFT between fetuses of both sexes was observed. DHT was not detectable in amniotic fluid. The results suggest the potential value of AFT for determining fetal sex in midtrimester pregnancy and confirm that maternal T and DHT increase during pregnancy and that cord T levels reflect fetal gonadal androgen synthesis.