Display omitted ► Studies on the mechanism of interactions between cells and nanoparticles (NPs) must be improved. ► We discuss the possibilities and pitfalls in studies of endocytic pathways followed by NPs. ► We evaluate the use of inhibitors, mutated proteins, siRNAs and colocalization experiments in such studies. ► We discuss intracellular transport, recycling to cell exterior, disturbance of cell functions, and metabolism of NPs. ► Knowledge about cellular fate of NPs is essential to develop nanoparticles for clinical use. During recent years there has been much interest in the use of nanoparticles for in vitro studies as well as for delivery of drugs and contrast agents in animals and humans. To this end it is necessary to increase our understanding of how these particles are taken up and transported within the cells, and to which extent they are metabolized and secreted. In this review we discuss the possibilities, challenges and pitfalls of studying endocytic pathways involved in cellular uptake of nanoparticles. Thus, the use of pharmacological inhibitors, expression of mutated proteins, use of siRNAs and colocalization experiments in such studies are critically evaluated. Although the main focus is on cellular uptake, also aspects of intracellular transport, recycling of nanoparticles to the cell exterior, disturbance of cellular functions, and metabolism of nanoparticles are discussed.