Background: Carbon monoxide (CO) poisoning is the leading cause of poisoning mortality and morbidity in the USA. Carboxyhemoglobin (COHb) levels are not predictive of severity or prognosis. At this time, the measurement of mitochondrial respiration may serve as a biomarker in CO poisoning. The primary objective of this study was to assess changes in mitochondrial function consisting of respiration and generation of reactive oxygen species (ROS) in peripheral blood mononuclear cells (PBMCs) obtained from patients with CO poisoning. Methods: PBMCs from patients having confirmed CO exposure treated with hyperbaric oxygen or HBO (CO group) and healthy controls (control group) were analyzed with high-resolution respirometry. PBMCs were placed in a 2-ml chamber at a final concentration of 3–4×10 6 cells/ml to simultaneously obtain both respiration and hydrogen peroxide (H2O2) production. In the CO group, we performed measurements before and after patients underwent their first HBO treatment. Results: We enrolled a total of 17 subjects, including 7 subjects with confirmed CO poisoning and 10 subjects in the control group. The CO group included five (71.4%) men and two (28.6%) women having a median COHb of 28%. There was a significant decrease in respiration as measured in pmol O2 ×s−1 ×10−6 PBMCs in the CO group (pre-HBO) when compared to the control group: maximal respiration (18.4±2.4 versus 35.4±2.8, P<0.001); uncoupled Complex I respiration (19.8±1.8 versus 41.1±3.8, P < 0.001); uncoupled Complex I+II respiration (32.3±3.2 versus 58.3±3.1, P<0. 001); Complex IV respiration (43.5±2.9 versus 63.6±6.31, P<0.05). There were also similar differences measured in the CO group before and after HBO treatment with an overall increase in respiration present after treatment. We also determined the rate of H2O2 production simultaneously with the measurement of respiration. There was an overall significant increase in the H2O2 production in the CO group after HBO treatment when compared to prior HBO treatment and the control group.