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Increasing exposure to nanoparticles (NPs) has raised concerns regarding their health and safety profiles in humans and animals, especially in developing organisms, which may display increased sensitivity to NP toxicity. The present study examined the effects of gestational exposure to carbon black NP (CB-NP) on the development of the offspring immune system. Pregnant mice were exposed to CB-NP (95μg/kg body weight) by intranasal instillation on gestational days 9 and 15. The thymus and spleen were collected from their offspring mice on postnatal day (PND) 1, 3 and 5. Thymocyte and splenocyte phenotypes were examined by determining the expression of cell-surface molecules using flow cytometry. Gene expression in the thymus and spleen was examined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Prenatal exposure to CB-NP increased total thymocytes and their immunophenotypes (CD4(-)CD8(-) and CD4(+)CD8(+) cells). It also induced an increase in total lymphocytes, and CD4(-)CD8(-), particularly CD3(-)B220(-)cells, at PND 5 in the spleen of newborn male offspring, reflecting the stimulation of immature splenocytes. Furthermore, mRNA expression of genes related to the induction of peripheral tolerance (i.e. thymic Traf6) was upregulated. These data suggest that respiratory exposure to CB-NP during middle and late gestation may have allergic or inflammatory effects in male offspring, and may provide initial information on the potential developmental immunotoxicity of nanoparticles.