Etimicin (ETM),a fourth-generation aminoglycosides (AGs),is now widely clinically used in China due to its high efficacy and low toxicity.However,the mechanisms underlying its low nephrotoxicity and ototoxicity remain unclear.In the present study we compared the antibacterial and toxicity profiles of etimicin,gentamicin (GM,a second-generation AG),and amikacin (AMK,a third-generation AG),and investigated their pharmacokinetic properties in the toxicity target organs (kidney and inner ear) and subcellular compartments.We first demonstrated that ETM exhibited superior antibacterial activities against clinical isolates to GM and AMK,and it exerted minimal nephrotoxicity and ototoxicity in rats following multi-dose administration.Then,we conducted pharmacokinetic studies in rats,showed that the three AGs accumulated in the kidney and inner ear with ETM being distributed to a lesser degree in the two toxicity target organs as compared with GM and AMK high-dose groups.Furthermore,we conducted in vitro experiments in NRK-52E rat renal tubular epithelial cells and HEI-OC1 cochlear hair cells,and revealed that all the three AGs were distributed predominantly in the mitochondria with ETM showing minimal accumulation;they not only directly inhibited the activity of mitochondrial complexes Ⅳ and Ⅴ but also inhibited mitochondrial function and its related PGC-1α-NRF1-TFAM pathway;ETM caused minimal damage to the mitochondrial complex and mitochondrial biogenesis.Our results demonstrate that the minimal otonephrotoxicity of ETM results from its lesser accumulation in mitochondria of target cells and subsequently lesser inhibition of mitochondrial function.These results provide a new strategy for discovering novel AGs with high efficacy and low toxicity.