The aim of this study was to test the influence of nanoparticle size and surface area (SA) on cytokine secretion by co-cultures of pulmonary epithelial cells (A549), macrophages (differentiated THP-1 cells) and endothelium cells (EA.hy926) in a two-compartment system. We used monodisperse amorphous silica nanoparticles (2, 16, 60 and 104nm) at concentrations of 5Î¼g/cm(2) cell culture SA or 10cm(2) particle SA/cm(2). A549 and THP-1 cells were exposed to nanoparticles for 24h, in the presence of EA.hy926 cells cultured in an insert introduced above the bi-culture after 12h. Supernatants from both compartments were recovered and TNF-Î±, IL-6, IL-8 and MIP-1Î± were measured. Significant secretion of all cytokines was observed for the 2nm particles at both concentrations and in both compartments. Larger particles of 60nm induced significant cytokine secretion at the dose of 10cm(2) particle SA/cm(2). The use of multiple cellular types showed that cytokine secretion in single cell cultures is amplified or mitigated in co-cultures. The release of pro-inflammatory mediators by endothelial cells not directly exposed to nanoparticles indicates a possible endothelium activation after inhalation of silica particles. This work shows the role of size and SA in cellular response to amorphous nanosilica.