Fetal extracellular DNA is mainly derived from apoptotic bodies of trophoblast. Recent studies have shown size differences between fetal and maternal extracellular DNA. We have examined the quantification of fetal (SRY gene) and total (GLO gene) extracellular DNA in maternal plasma in different fractions (100–300, 300–500, 500–700, 700–900, and >900 bp) after size fractionation by agarose gel electrophoresis. DNA was extracted from maternal plasma samples from 11 pregnant women carrying male foetuses at the 16th week of gestation. Fetal circulatory DNA was mainly detected in the 100–300 bp fraction with the median concentration being 14.4 GE/ml. A lower median amount of 4.9 GE/ml was also found in the 300–500 bp fraction. Circulatory DNA extracted from the 100–300 bp fraction contained 4.2 times enriched fetal DNA when compared with unseparated DNA sample. Fetal DNA within the 300–500 bp fraction was 2.5 times enriched. Circulatory fetal DNA is predominantly present in a fraction with molecular size <500 bp, which can be used for the detection of paternally inherited alleles. However, the usage of size-separated DNA is not suitable for routine clinical applications because of risk of contamination.