The reliability of the polymerase chain reaction (PCR) for prenatal diagnosis of human cytomegalovirus (HCMV) infection was determined by retrospective testing of 35 amniotic fluids identified previously as positive or negative for HCMV by virus isolation. Amniocentesis was performed in 26 pregnant women with primary HCMV infection at 14-36 weeks gestation, 3-21 weeks after maternal infection. Blood samples were obtained from 20 fetuses for IgM determination and/or virus isolation. Amniotic fluid culture led to antenatal diagnosis of HCMV in 9 of the 13 infected fetuses (sensitivity 69.2%) with one case diagnosed at a second sampling. PCR was able to detect one additional infected fetus (10/13, sensitivity 76.9%). Nested PCR did not increase sensitivity of prenatal diagnosis. Three cases were not diagnosed by all the techniques employed. The specificity of virus isolation from and DNA detection by PCR in amniotic fluid was 100%. The negative predictive value for virus isolation from amniotic fluid was 76.5% and for DNA detection by PCR 81.2%, whereas the positive predictive value was 100% for both techniques. The results showed that neither approach can detect all cases of congenital HCMV infection prenatally, and that the time interval between maternal infection and sampling seems to be a major factor affecting the reliability of prenatal diagnosis.