To estimate whether potential clinical applications of cell-free fetal and total DNA in the field of noninvasive prenatal diagnosis need to be adjusted for maternal smoking status. In this study, using 344 maternal blood samples from the second trimester of pregnancy, circulating cell-free DNA in maternal plasma samples, specific for the SRY and DYS14 loci (representing fetal DNA) and GAPDH sequence (representing total genomic DNA) were quantified by real-time polymerase chain reaction. Fetal sex determination was 100% accurate using a combination of probes for SRY and DYS14. The levels of DYS14 and SRY detected were significantly correlated (r=0.884, P<.001). No significant difference was seen between the quantitative levels of cell-free male fetal DNA between the smoking groups and control group. Similarly, no significant difference was seen in the amount of total cell-free DNA in the study population. In contrast to first- and second-trimester screening assays for Down syndrome, where smoking status significantly affect levels of maternal serum analytes, smoking status does not affect quantitative levels of cell-free fetal DNA or total cell-free DNA in maternal plasma. II.