Phthalates are used in plastics to promote flexibility, but as they easily migrate from the plastic humans are widely exposed. The exposure to phthalates has been associated with worsening and incidence of allergic diseases like asthma in epidemiological studies. This suggestion is partly supported by experimental studies showing that phthalates can induce adjuvant and immunological effects in mice and cell models. The hypothesis of this study was that the model phthalate di-n-butyl phthalate (DBP) affects systemic and pulmonary primary immune cells after in vitro exposure to concentrations relevant for human exposure levels, and that different cell models would vary in their sensitivity to DBP. Three experimental models were used to study the effects of 0.01-100µM DBP; a whole blood assay (WBA) relevant for systemic exposure, and sputum and monocyte-derived macrophages with relevance for inhalation exposure. Three inflammatory stimuli were used in combination with DBP: the toll like receptor (TLR) agonists lipopolysaccharide (LPS) or R848, or a combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (P/I), activating T cells. The release of inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA) and/or by the cytometric bead assay (CBA) using flow cytometry. The effects of DBP were generally greater after a challenge with inflammatory stimuli for the last 4h of the 24h DBP-exposure, when compared to combined exposure for 24h. DBP caused U-shaped response curves for P/I-induced cytokine release in whole blood, for tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-5, with a 30-45% reduction at 0.01µM -10µM DBP. This reduction was possibly linked to a parallel increase in cytotoxicity. The effects of DBP in combination with the other inflammatory stimuli in whole blood were less pronounced. For sputum macrophages, LPS-induced cytokine release also showed a U-shaped decrease for 0.01-10µM DBP, but only for TNFα. DBP did not affect the cytokine release from monocyte-derived macrophages. In conclusion, environmentally relevant concentrations of DBP decreased the release of cytokines from both innate and adaptive immune cells in vitro, suggesting that DBP exposure may lead to an impaired immune response. Moreover, cytokine release from whole blood after P/I stimulation and LPS-induced TNFα release from sputum macrophages were more sensitive to DBP exposure than the other model systems and endpoints.