Human amniotic fluid samples obtained from normal pregnancies and supernatants of normal full-term placental and decidual tissues were assayed in a specific radioimmunoassay (RIA) and in a sensitive bioassay for tumor necrosis factor (TNF). TNF was detected in 91% of amniotic fluid samples and in all placental and decidual supernatants assayed by the RIA. Amniotic fluid samples collected during the second trimester of pregnancy contained significantly higher concentrations of TNF than samples collected during the third trimester, average concentrations being 2.5 ng/ml and 0.9 ng/ml, respectively. Parallel testing in the bioassay showed that most of the amniotic fluid TNF is biologically inactive, whereas placental and decidual supernatants had biologic activity correlating with TNF levels detected by the RIA. The quantities of TNF detected by the RIA ranged from 1.1 to 2.8 ng/ml in placental supernatants and from 3.9 to 8.5 ng/ml in decidual supernatants. The presence of TNF in normal amniotic fluids and in placental and decidual supernatants suggest that this cytokine has a physiologic role in human pregnancy.