To examine whether concentrations of free extracellular fetal circulatory DNA in maternal plasma are stable or fluctuate. Consecutive blood samples were drawn from 13 healthy nonpregnant volunteers and from 16 healthy pregnant women over 3 days. DNA was isolated from the plasma fraction and quantified by real-time polymerase chain reaction (PCR). In nonpregnant controls the total amount of cell free DNA fluctuated by an average of 13.5-fold. In samples obtained from pregnant women the amount of maternal cell free DNA varied by an average of 21.5-fold. Because ten of those women were pregnant with male fetuses, the concentration of free fetal DNA in these cases was determined by a real-time PCR assay for the Y chromosome. The mean variation in free fetal DNA levels in male fetuses was 2.2-fold. The degree of variation in free fetal DNA concentrations observed in this study was similar to published values, so these results imply that care should be exercised when considering quantitation of this fetal material for potential diagnostic or screening purposes.