The increasing number of nanotechnology products on the market poses increasing human health risks by particle exposures. Adverse effects of silver nanoparticles (AgNPs) in various cell lines have been measured based on exposure dose after a fixed time point, but NP uptake kinetics and the time-dependent internal cellular concentration are often not considered. Even though knowledge about relevant timescales for NP uptake is essential, e.g. for time- and cost-effective risk assessment through modelling, insufficient data are available. Therefore, the authors examined uptake rates for three different AgNP sizes (20, 50 and 75 nm) and two tissue culture medium compositions (with and without foetal calf serum, FCS) under realistic exposure concentrations in pulmonary epithelial 16HBE14o-cells. The quantification of Ag in cells was carried out by high-resolution inductively coupled plasma mass spectrometry. We show for the first time that uptake kinetics of AgNPs into 16HBE14o-cells was highly influenced by medium composition. Uptake into cells was higher in medium without FCS, reaching approximately twice the concentration after 24 h than in medium supplemented with FCS, showing highest uptake for 50-nm AgNPs when expressed on a mass basis. This optimum shifts to 20 nm on a number basis, stressing the importance of the measurand in which results are presented. The importance of our research identifies that not just the uptake after a certain time point should be considered as dose but also the process of uptake (timing) might need to be considered when studying the mechanism of toxicity of nanoparticles.