Early pregnancy factor (EPF), an immunosuppressive substance, appears in mammalian maternal serum at a very early stage after fertilization. To investigate the normal distribution of EPF in the human body, the rosette inhibition test has been used to detect EPF in human amniotic fluid. The mean value of rosette inhibition titer (RIT) was significantly higher (5.93 +/- 0.43 SE) in 52 amniotic fluid samples (16-30 weeks' gestation) than in 23 nonpregnant sera (3.54 +/- 0.11), indicating that something in amniotic fluid inhibits rosette formation. This inhibitory factor is recognized as EPF or EPF-like substance for its activity significantly correlated with that of EPF in serum from the same donor (r = 0.756: P less than 10(-6]. The immunological significance of the EPF-like substance in amniotic fluid may be important to the survival of semiallogeneic conceptus.