We describe the development of a nested polymerase chain reaction (PCR) technique used to detect human parvovirus B19 DNA. It was performed in a two-step reaction, first with a pair of outer primers, then with a pair of inner (ie, nested) primers. Primer sequences were selected in the VP1 gene, corresponding to the capsid protein, of human parvovirus B19. To establish the nested PCR assay, the plasmid pGEM-1 containing almost the entire coding sequence of a human parvovirus B19 isolate was used. The nested PCR could detect up to 1.8 x 10(-3) ag of B19 DNA, equivalent to 10(-4) genomes, by electrophoresis. No amplification product was detected by gel electrophoresis when the reaction mixture contained human placental DNA, cytomegalovirus DNA, and sterile distilled water as templates. We used this assay to evaluate four cases of maternal B19 infection that were diagnosed by determination of the presence of anti-B19 immunoglobulin-M in maternal serum. The advantages of our nested PCR for detecting B19 DNA are plating simplicity, safety, sensitivity, and specificity. Our results suggest that this method may have general applicability in the evaluation of nonimmune hydrops fetalis and in the documentation of the natural history of fetal infection with B19 when applied to specimens of amniotic fluid or fetal blood.