The purpose of this study was to determine the proportion of fetal nucleated red blood cells (NRBCs) among enriched NRBCs and to evaluate the effectiveness of enriching NRBCs in maternal blood using fluorescence-activated cell sorting (FACS) to separate NRBCs. The origin of enriched NRBCs was determined using fluorescence in situ hybridization (FISH) methods. Y-specific signals were observed in 4.6 +/- 1.5 per cent of the enriched cells from 14 of 16 (87.5 per cent) pregnant women who gave birth to boys. In this series, the specificity of the fetal sex diagnosis was 100 per cent, the sensitivity 88 per cent, and the negative predictive value 86 per cent. Fetal NRBCs are present in maternal blood and FACS has the potential to enrich fetal NRBCs. Fetal cells were estimated to be enriched more than 10,000-fold in the first trimester and more than 100-fold in the third trimester. Average frequencies of fetal cells in maternal blood were 8.1 x 10(-5) and 1.6 x 10(-5) in the first trimester and the second/third trimesters. However, most of the NRBCs in maternal blood are maternal in origin.