Nanotechnology applications already on the market or in development promise great benefits for humans as well as the environment. Simultaneously, the pressure to advance the development of fast methods for evaluating the potential risks of increased human exposure to nanomaterials is augmented. One way forward would be to enhance the role of in vitro toxicity studies in risk assessment procedures of nanomaterials. However, to maximize the use of in vitro assays for this purpose, their values and limitations need to be revealed. Even in risk assessment frameworks for regular chemicals, in vitro studies play a minor role. A comparative analysis of published in vitro data with nanomaterials demonstrates that there are a number of issues that need resolving before in vitro studies can play a role in the risk assessment of nanomaterials.