Due to the existence of considerable quantities of metallic and carbonaceous impurities, the key factor and mechanism for the reported toxicity of carbon nanotubes (CNTs) are unclear. Here, we first quantify the contribution of metal residues and fiber structure to the toxicity of CNTs. Significant quantities of metal particles could be mobilized from CNTs into surrounding fluids, depending on the properties and constituents of the biological microenvironment, as well as the properties of metal particles. Furthermore, electron spin resonance measurements confirm that hydroxyl radicals can be generated by both CNTs containing metal impurities and acid-leachable metals from CNTs. Several biomolecules facilitate the generation of free radicals, which might be due to the participation of these biomolecules in redox cycling influenced by pH. Among several major metal residues, Fe has a critical role in generating hydroxyl radicals, reducing cell viability and promoting intracellular reactive oxidative species. Cell viability is highly dependent on the amount of metal residues and iron in particular, but not tube structure, while the negative effect of CNTs themselves on cell viability is very limited in a certain concentration range below 80 lg ml À1 . It is crucial to systematically understand how these exogenous and endogenous factors influence the toxicity of CNTs to avoid their undesirable toxicity. Keywords: biological microenvironments; carbon nanotube; metal impurities; reactive oxygen species; toxicity INTRODUCTION Engineered nanomaterials possess novel properties and have been used worldwide in numerous consumer products, for example, food, clothes, pharmaceuticals and cleaning products. Estimates suggest that by 2015 nanotechnology will have a trillion-plus dollar global economic impact. 1 Therefore, ensuring the safety of nanomaterials is of great importance to the tremendous commercial applications of nanotechnology. Safety evaluation of nanomaterials should consider their behaviors in various aspects, including their interaction with proteins, DNA, lipids, membranes, organelles, cells, tissues, biological fluids and even the dissolution of metal constituents. [2][3][4] Carbon nanotubes (CNTs) have attracted great interest from both scientists and the industry because their high aspect ratios, strength and remarkable physical properties make them a unique material with a wide range of promising applications. Many applications of CNTs in biomedicine have been proposed, including nanoelectronics, 4 biosensors, 5 biomolecular recognition devices and molecular transporters, 6 artificial water channels, 7 cancer therapy 8,9 and photoacoustic molecular imaging. 10 Because transitional metals are often used as catalysts during CNT synthesis, 11 it is unavoidable that as-received CNTs are contaminated by catalyst residues. 12 The catalyst precursor (such as iron carbonyl) decomposes, and nanometer-sized metal particles form from the decomposition products. Therefore,