Two pregnant women with secondary syphilis underwent amniocentesis and evaluation for fetal syphilis. In both cases, motile spirochetes, typical of Treponema pallidum, were observed during dark-field microscopic examination of the amniotic fluid. The presence of T pallidum was confirmed by antitreponemal monoclonal antibody immunofluorescence assays and by rabbit infectivity tests using the amniotic fluid. In the first case, an infant at 35 weeks' gestation delivered within 24 hours of amniocentesis had hepatosplenomegaly, osteochondritis, and neurosyphilis. In the second case, a fetus at 24 weeks' gestation was hydropic and a fetal blood sample showed anemia, thrombocytopenia, and elevated liver enzymes. Fetal syphilis was confirmed by rabbit infectivity testing using fetal blood obtained by funipuncture. This is the first report of the diagnosis of fetal syphilis by funipuncture and confirmation of the presence of virulent T pallidum in the blood of a human fetus. The mother was treated for secondary syphilis, but the infant had residual signs of congenital infection at birth 14 weeks later. Neonatal serum from the first case and fetal serum from the second case showed specific immunoglobulin M reactivity with the 47-kd antigen of T pallidum by Western blot assays. A new wild-type strain of T pallidum, designated DAL-1, was isolated from the amniotic fluid of the first case and is available for future studies. We conclude that the presence of T pallidum in amniotic fluid or fetal blood indicates fetal-placental infection. Further investigation is necessary to determine the pathogenesis of amniotic fluid infection and its role in the prenatal diagnosis of congenital syphilis.