The enrichment of fetal erythroblasts from the peripheral blood of pregnant women is currently actively pursued for the development of a non-invasive means of prenatal diagnosis. Since erythroblasts in maternal blood are not all of fetal origin, and currently no reliable method exists to distinguish between the maternal and fetal erythroblasts, their use for prenatal diagnosis is not without uncertainty. The purpose of this study was to determine the percentage of fetal erythroblasts in maternal blood at the single cell level and to what extent such cells can reproducibly be used for polymerase chain reaction (PCR)-based prenatal diagnostic analyses. Erythroblasts were enriched from the peripheral blood of rhesus negative pregnant women using magnetic cell sorting (MACS). Single erythroblasts identified morphologically were individually micromanipulated and analysed by a multiplex PCR reaction for the fetal SRY and rhesus D genes. As a control for the PCR reaction the beta-globin gene was used. The PCR results were validated by the results obtained by invasive procedures. In all instances where single erythroblasts were examined, the correct fetal genotype for the two fetal specific loci was detected. Furthermore, our results indicate that approximately 50% of the enriched erythroblasts are of fetal origin.