The increased application of carbon nanotubes (CNTs) has raised the level of public concern regarding possible toxicities. Using in vitro cellular assays, we were able to assess the immunotoxicity of pristine multi-wall carbon nanotubes (MWCNTs) and their derivatives, covalently functionalized with carboxyl (COOH) or polyethylene glycol (PEG), in rodent macrophage cells. Moreover, special focus was placed on the role of surface modification and nanotubes aggregation on toxicity. Results showed that pristine MWCNTs reduce cell viability compared with functionalized MWCNTs in RAW 264.7 macrophages when incubated at concentrations of 25, 50, 100, 200, 400, and 800 mu g/mL. However, in addition to causing cytotoxicity, functionalized MWCNTs induce serious inflammatory responses, as indicated by the production of inflammatory cytokines including TNF-alpha, IL-1 beta and IL-6 at various MWCNTs concentrations (25, 50, 100, and 200 mu g/mL). Particle surface modification and dispersion status in biological medium were key factors in determining cytotoxicity. These findings imply that MWCNTs-induced inflammatory responses in macrophages may be associated with surface modification and aggregation of MWCNTs, which is reflected by alteration of inflammatory cytokine expression. (C) 2012 Elsevier B.V. All rights reserved.