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Silver nanoparticles (AgNPs) show a plethora of possible applications due to their antimicrobial properties. Different coatings of AgNPs are used in order to increase stability, availability, and activity. However, the question about the toxicity after prolonged exposure still remains. Here, we show that different surface coatings affect in vitro toxicity and internalization of AgNPs in porcine kidney (PK15) cells. AgNPs coated with cetyltrimethylammonium bromide (CTAB), poly(vinylpyrrolidone) (PVP), sodium bis(2-ethylhexyl)-sulfosuccinate (AOT), poly-L-lysine (PLL), and bovine serum albumin (BSA) were toxic at the concentration of 10 mg Ag/L and higher. The toxicity increased in the following manner: PVP-AgNPs < CTAB-AgNPs < PLL-AgNPs < AOT-AgNPs < BSA-AgNPs. All types of AgNPs were internalized by the PK15 cells in a dose-dependent manner with greater internalization of AgNPs bearing positive surface charge. Transmission electron microscopy (TEM) experiments showed that AgNPs were located in the lysosomal compartments, while the co-treatment with known inhibitors of endocytosis pathways suggested macropinocytosis as the preferred internalization pathway. When inside the cell, all types of AgNPs induced the formation of reactive oxygen species while decreasing the concentration of the cell's endogenous antioxidant glutathione. The comet assay indicated possible genotoxicity of tested AgNPs starting at the concentration of 2 mg Ag/L or higher, depending on the surface functionalization. This study demonstrates the toxicity of AgNPs pointing to the importance of biosafety evaluation when developing novel AgNPs-containing materials., (© 2022 John Wiley & Sons Ltd.)