The presence of various genomic sequences in the pool of extracellular DNA was studied by cloning and sequencing the DNA eluted from the surface of HeLa cells. Sequences of 19 genes, 10 pseudogenes, and 41 repeated elements were found in 103 clones. Sequences of LINE repeats were found in 17% of the clones; consequently real-time PCR assay for quantification of LINE repeats was chosen to study the influence of protein transport inhibitors on the concentration of free and cell-surface-bound DNA in the cultures of HUVEC and HeLa cells. Treatment of both cell lines with the inhibitors did not interfere with the concentration of extracellular DNA in the growth medium, except for chloroquine, which doubled the concentration of extracellular DNA in HUVEC culture. The treatment of HUVECs with monensin, glyburide, or methylamine decreases the cell-surface-bound DNA concentration by 30, 35, and 19%, respectively. The incubation of HeLa cells with monensin reduces the concentration of cell-surface-bound DNA by 15%; however, the treatment with glyburide increases cell-surface-bound DNA concentration by 50%. The data obtained demonstrate the involvement of vesicular transport in generation of extracellular DNA.