We examined whether the 33-kDa serine protease prostate-specific antigen (PSA) is present in amniotic fluid and, if so, whether its concentration changes with gestational age. Analyzing 115 amniotic fluids with a highly sensitive immunofluorometric procedure, we found PSA in all the amniotic fluids examined and established that its concentration increases with increasing gestational age from 11 to 21 weeks, decreasing at delivery. PSA in amniotic fluid is present predominantly in the free (33 kDa) form; a minor fraction (< 20%) is present bound to alpha 1-antichymotrypsin. No significant correlation was seen between PSA and alpha-fetoprotein (AFP) in amniotic fluid or maternal serum in samples with high AFP. Amniotic fluid PSA was also measurable by two different established methods for PSA. Pregnant women had higher concentrations of serum PSA than nonpregnant women. The highest PSA concentration in amniotic fluid was associated with a pregnancy that was complicated by the Rhesus incompatibility syndrome but the source of the PSA was not established. From recent literature reports, and the association of PSA with prostate and breast tumors, we think PSA may serve as a growth factor regulator in cancer and in normal fetal development during pregnancy.