ORganically MOdified SILica (ORMOSIL) nanoparticles (NPs) appear promising carriers for the delivery of drugs to target tissues but concerns on possible cytotoxic effects exist. Here, we studied the in vitro responses to ORMOSIL NPs in different types of human lung cells to determine the effects of polyethylene glycol (PEG) coating on NP cytotoxicity. Non-PEG NPs caused a concentration-dependent decrease of viability of all types of cells, while PEG NPs induced deleterious effects and death in carcinoma alveolar type II A549 cells but not in CCD-34Lu fibroblasts and NCI-H2347 adenocarcinoma cells. Reactive oxygen species were detected in cells incubated with PEG NPs, but their deactivation by superoxide dismutase and catalase did not protect A549 cells from death, suggesting that the oxidative stress was not the main determinant of cytotoxicity. Only in A549 cells PEG NPs modulated the transcription of genes involved in inflammation, signal transduction and cell death. Transmission electron microscopy evidenced a unique intracellular localization of PEG NPs in the lamellar bodies of A549 cells, which could be the most relevant factor leading to cytotoxicity by reducing the production of surfactant proteins and by interfering with the pulmonary surfactant system.