OBJECTIVE: To develop a noninvasive method suitable for clinical prenatal diagnosis. STUDY DESIGN: Fetal nucleated erythrocytes were separated from peripheral blood of 17 healthy pregnant women using small magnetically activated cell sorting columns (MiniMACS) following density gradient centrifugation and dual antibody labeling methods. The protocol was designed to compare the efficacy of antitransferrin receptor (CD71)/antiglycophorin A (GPA) antibodies with antithrom-bospondin receptor (CD36)/anti-GPA antibodies in identifying nucleated erythrocytes in maternal blood. Cytospin preparations of the isolated cells were subjected to in situ hybridization with specific DNA probes for the Y chromosome and chromosome 21 to confirm the fetal origin. RESULTS: After MiniMACS the enrichment factors for the CD71/GPA- and CD36/GPA-positive cells from maternal blood were similar, and the percentages of fetal cells recovered did not differ. Seven of seven male pregnancies were correctly identified. One case of trisomy 21 was detected. CONCLUSION: The in situ hybridization analysis of fetal nucleated erythrocytes isolated from maternal blood using single density gradient centrifugation, anti-CD71/anti-GPA immunostaining and MiniMACS could be an accurate, sensitive and noninvasive method for prenatal diagnosis.