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Background: Early in life, natural exposure to microbial components (eg, endotoxin) may mitigate allergy and asthma development in childhood. Bacterial DNA is a potent stimulus for the innate immune system; its immune stimulatory potential in dust is unknown. Objectives: We sought to quantify bacterial DNA and endotoxin content in dust from urban homes, rural homes, farm homes, and farm barns and to determine if dust DNA is immune-stimulatory. Methods: Total DNA, bacterial DNA, and endotoxin were measured in 32 dust samples. To measure bacterial DNA content, a quantitative polymerase chain reaction assay specific for bacterial ribosomal DNA was developed. Peripheral blood mononuclear cells from 5 adults were stimulated with endotoxin-free dust DNA with/without lipopolysaccharide (LPS) from selected dust samples. IL-12p40, IL-10, and tumor necrosis factor-α were measured in cell supernatants by enzyme-linked immunosorbent assay. Results: Bacterial DNA in dust correlated with endotoxin (r = 0.56, P < .001) and total DNA content (r = 0.51, P = .003). The highest bacterial DNA levels were measured in farm barns (mean, 22.1 µg/g dust; range, 1.3 to 56.2), followed by rural homes (6.3 µg/g; 0.2 to 20), farm homes (2.2 µg/g; 0.1 to 9.1), and urban homes (0.6 µg/g; 0.1 to 1.2). Farm barn DNA significantly potentiated (P ≤ .05) LPS-induced IL-10 and IL-12 p40 but not tumor necrosis factor-α release (13-fold, 3-fold, and 1.5-fold increases, respectively). DNA from 6 urban homes did not demonstrate this LPS-potentiating effect. Conclusions: Endotoxin is a marker for bacterial DNA, which is also higher in locales of lower asthma and allergy prevalence. DNA from farm barn dust augments the immune modulatory effects of endotoxin and may combine with exposure to other such naturally occurring microbial components to mitigate allergy and asthma development. (