Despite the emerging sense that well-engineered, novel-property nanomaterials will continue to be studied and will be utilized to a greater extent in the future for a wide range of biomedical applications, there is also a growing concern that unusual and unexpected toxicity may arise from desired properties, such as higher reactivity, unusual complex shapes, and accessibility to cells, which man-made materials possess in the nanoscale [1–14]. Therefore, it is essential to establish a new toxicology study that deviates from or modifies conventional methods. It is difficult to adequately assess the biological effects of nanoparticles due to the fact that their cellular uptake, in vivo behavior and excretion are controlled by numerous factors. Any change in the composition, size, morphology, route of exposure or dosage can significantly affect the immunological response and toxic effects elicited by the nanoparticle. Additionally, these properties can affect the outcome of a particular cytotoxicity or toxicological assay due to unforeseen interferences and inadequate controls. In this respect, this special focus issue of Nanomedicine will provide the chance to read and discuss the most recent and significant research efforts toward nanotoxicology in the aspects of new methods, biological models, and experimental concerns. Drezek and colleagues have provided an extensive review on the current status of nanoparticle biodistribution and toxicity studies [15]. The authors discuss the need for long-term in vivo studies to fully investigate the toxicity, immune response and excretion over time of nanoparticles, and the need for a more systematic approach toward in vitro experiments for improved correlation between nanoparticle properties and observed effects. To address the safety standards of new emerging nanotechnology, a review by Krug and colleagues discusses the current reliability of in vitro toxicity assays as well as major problems and challenges of assay performance and validation in which the group recommends a series of controls to improve experimental quality that will increase the safe and sustainable use of nanotechnology for the future [16]. In addition, a review by Lee and colleagues highlights the most significant factors affecting nanoparticle cytotoxicity assays and the improved experimental considerations needed to address them in order to gain reliable data and better define the adverse effects of nanoparticles [17]. A review by Porter and colleagues discusses the challenges associated with characterizing the toxicity of carbon nanotubes and the need for complementary nanometrology to relate their physiochemical properties with their toxicity [18]. The authors suggest a combination of high resolution electron microscopy techniques and cell viability assays to understand cytotoxic mechanisms of the targeting site and stability of carbon nanotubes inside cells and tissues. With regard to specific classes of nanomaterials, Fubini discusses the various types of high-aspect ratio nanoparticles, focusing on the most relevant characteristics that lead to their toxic effects [19]. The author states that similarities in form or composition are not adequate to establish toxicity and that the effect of size, form, durability, composition and surface modification must be investigated for all high-aspect ratio nanoparticles. Choy and Choi provide an overview of the cellular uptake mechanism, behavior and toxicity of layered double hydroxides both in vitro and in vivo, focusing on the effect of physiochemical properties, dosage and interaction with biological systems on biocompatibility [20]. A research article by Zhang, Baumer and Monteiro-Riviere explores the toxicity and cellular uptake of quantum dots by dendritic cells, shedding new light on the dependence of cellular uptake pathways on cell type and cell differentiation [21]. Garcia-Bennett gives an overview of ordered mesoporous nanomaterials for use in “It is essential to establish a new toxicology study that deviates from or modifies conventional methods.” SPECIAL FOCUS y Nanotoxicology: Materials & Methods