The objective of the present study was to determine the mitochondrial toxicity mechanisms of linezolidrelated hyperlactatemia. Five patients on a long-term schedule of linezolid treatment were studied during the acute phase of hyperlactatemia and after clinical recovery and lactate normalization following linezolid withdrawal. Mitochondrial studies were performed with peripheral blood mononuclear cells and consisted of measurement of mitochondrial mass, mitochondrial protein synthesis homeostasis (cytochrome c oxidase [COX] activity, COX-II subunit expression, COX-II mRNA abundance, and mitochondrial DNA [mtDNA] content), and overall mitochondrial function (mitochondrial membrane potential and intact-cell oxidative capacity). During linezolid-induced hyperlactatemia, we found extremely reduced protein expression (16% of the remaining content compared to control values [100%], P < 0.001) for the mitochondrially coded, transcribed, and translated COX-II subunit. Accordingly, COX activity was also found to be decreased (51% of the remaining activity, P < 0.05). These reductions were observed despite the numbers of COX-II mitochondrial RNA transcripts being abnormally increased (297%, P ؍ 0.10 [not significant]) and the mitochondrial DNA content remaining stable. These abnormalities persisted even after the correction for mitochondrial mass, which was mildly decreased during the hyperlactatemic phase. Most of the mitochondrial abnormalities returned to control ranges after linezolid withdrawal, lactate normalization, and clinical recovery. Linezolid inhibits mitochondrial protein synthesis, leading to decreased mitochondrial enzymatic activity, which causes linezolid-related hyperlactatemia, which resolves upon discontinuation of linezolid treatment.Linezolid belongs to a family of antibiotics (oxazolidinones) that inhibit bacterial protein synthesis by binding to 23S rRNA in the large ribosomal subunit and preventing the fusion of 30S and 50S ribosomal subunits and the formation of the translation initiation complex (1). It has shown excellent efficacy against gram-positive cocci, including Staphylococcus aureus, coagulase-negative staphylococci, enterococci, and streptococci, with MICs ranging from 0.5 to 4 mg/liter (16). Furthermore, linezolid has 100% oral bioavailability and reaches high concentrations at different sites (skin, synovial fluid, bone, cerebrospinal fluid, or lung), thus being a good alternative for the long-term treatment of orthopaedic implant infections, ventriculo-peritoneal shunts, and other infections related to foreign bodies in which gram-positive cocci are the main pathogens.