In the present work, we investigated the potential effects of Wi-Fi exposure on the developing immune system of young mice. Pregnant C57BL/6 mice were exposed or sham-exposed to a 2.45 GHz Wi-Fi signals. We evaluated the effects on immature immunity of mice by assessing splenocytes phenotype and functionality. The results of this study will provide useful data for health risk assessment by WHO related to RF fields. Pregnant C57 BL/6 mice, obtained 3 days post coitum were acclimated for 4 days. Dams and newborn mice were exposed or sham exposed at three SAR levels (whole-body specific absorption rate: 0.08, 0.4, and 4 W/kg) in a reverberation chamber. This free-running exposure system, specially designed for this study, emits a Wi-Fi signal in a cubic chamber with 6 antennas and mixing of the modes for a symmetrical and uniform exposure of the animals. Daily exposure lasted 2 hours. The groups were exposed for 2 weeks in utero and 5 weeks after birth for a total of 7 weeks of exposure. We performed three series of exposure. All experiments were performed blind. Temperature and humidity were controlled during the experiments. At the end of exposure, the 5-week-old mice were sacrificed and spleen collected. Single-cell suspensions were obtained after mechanical dissociation, isolation using a 40 ¹m cell strainer, and spleen erythrocyte lysis. Natural Killer (NK) cell isolation was performed using magnetic cell separation. Briefly, NK cells were labelled with specific monoclonal antibodies conjugated to paramagnetic particles. NK cells were separated from T and B-cells using a column. YAC-1 cells derived from mouse lymphoma (ECACC, Salisbury, UK) were used as target cells for the NK cytotoxicity assay. The number of splenocytes and the cell sub-populations distribution were determined. Evaluation of CD45, CD3, CD4, CD8, CD19, and NK1.1 expression was done by flow cytometry analysis allowing for comparison between exposed and sham-exposed splenocyte phenotypic profiles. We also performed ex-vivo stimulation of T and B cells using specific monoclonal antibodies (anti-CD3 and anti-CD28) or LPS for testing their proliferation ability. The functionality of splenocytes was also tested through evaluation of cytokine production (IL-2, TNF and IFN-γ) and expression of activation markers (CD25 and CD69). Finally, NK cell cytotoxic activity was analyzed using flow cytometry. This test was based on YAC-1 target cell labelling with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) and subsequent DNA-labelling with 7AAD for identifcation of target cells with compromised cell membranes. The results are expressed as percentage of dead targets on a cell-to-cell basis. The ongoing analysis of sub-population phenotype, functionality and responsiveness of lymphocytes will be completed and presented at the meeting.