Hypothesis: The surface chemistry of synthetic amorphous silicas is essential for their applicational performance and for understanding their interactions with biological matter. Synthesis of silica by flame spray pyrolysis (FSP) allows to control the content and type of hydroxyl groups which also affects the cytolytic activity. Experiments: By controlling the FSP process variables, silica nanoparticles with the same specific surface area but different surface chemistry and content of internal silanols are prepared by combustion of hexamethyldisiloxane sprays, as characterized by Raman and infrared spectroscopy, thermogravimetric analysis, and titration with lithium alanate. Cytolytic activity is assessed in terms of membrane damage in human blood monocytes in vitro. Findings: Unlike commercial fumed silica, FSP-made silicas contain a significant amount of internal silanol groups and a high surface hydroxyl density, up to similar to 8OH/nm(2), similar to silicas made by wet chemistry. Increasing the residence time of particles at high temperature during their synthesis reduces the internal and surface hydroxyl content and increases the relative amount of isolated silanols. This suggests incomplete oxidation of the silica matrix especially in short and "cold" flames and indicates that the silica particle formation pathway involves Si(OH)(4). The surface chemistry differences translate into lower cytolytic activity for "cold-" than "hot-flame" silicas. (C) 2017 Elsevier Inc. All rights reserved.