As noble metal nanoclusters (NCs) are widely employed in nanotechnology, their potential threats to human and environment are relatively less understood. Herein, the biological effects of ultra-small silver NCs coated by bovine serum albumin (BSA) (Ag-BSA NCs) on isolated rat liver mitochondria were investigated by testing mitochondrial swelling, membrane permeability, ROS generation, lipid peroxidation and respiration. It was found that Ag-BSA NCs induced mitochondrial dysfunction via synergistic effects of two different ways: (1) inducing mitochondrial membrane permeability transition (MPT) by interacting with the phospholipid bilayer of the mitochondrial membrane (not with specific MPT pore proteins); (2) damaging mitochondrial respiration by the generation of reactive oxygen species (ROS). As far as we know, this is the first report on the biological effects of ultra-small size nanoparticles (similar to 2 nm) at the sub-cellular level, which provides significant insights into the potential risks brought by the applications of NCs. It would inspire us to evaluate the potential threats of nanomaterials more comprehensively, even though they showed no obvious toxicity to cells or in vivo animal models. Noteworthy, a distinct toxic mechanism to mitochondria caused by Ag-BSA NCs was proposed and elucidated. (C) 2016 Elsevier B.V. All rights reserved.