This report describes the first identification and characterization of three chromosome-21-specific DNA sequences (and reference sequences from other chromosomes) that are differentially methylated between peripheral blood and placental tissue, with the aim of providing epigenetic biomarkers for quantifying cell-free fetal DNA in maternal plasma. To select sequences to be screened for differential methylation, three strategies were adopted: (i) investigating promoters of highly differentially expressed genes; (ii) choosing 'random' promoter regions; and (iii) choosing 'random' non-promoter regions. Over 200 pre-selected DNA sequences were screened using a methylation-specific restriction enzyme assay. Differentially methylated sequences located at 21q22.3 (AIRE, SIM2 and ERG genes), 1q32.1 (CD48 gene and FAIM3 gene), 2p14 (ARHGAP25 gene) and 12q24 (SELPLG gene) were identified. Bisulphite conversion confirmed that CpG sites within the AIRE promoter region are highly differentially methylated, and optimized methylation-specific primers for this region that are highly specific for placental DNA were devised. Next, it was shown that the methylation status of chorionic villus sample DNA from first trimester pregnancies matched the hypermethylated state of term placenta. Thus there is no indication of a difference in methylation status between early and term pregnancy for the sequences tested. The identified sequences constitute candidate biomarkers for non-invasive prenatal diagnosis of Down syndrome.