Given that the physiology of heme oxygenase-1 (HO-1) encompasses mitochondrial biogenesis, we tested the hypothesis that the HO-1 product, carbon monoxide (CO), activates mitochondrial biogenesis in skeletal muscle and enhances maximal oxygen uptake (VO₂max) in humans. In ten healthy subjects, we biopsied the vastus lateralis and performed max tests followed by blinded randomization to air or CO breathing (1hour/day at 100ppm for 5 days), a contralateral muscle biopsy on day 5, and repeat max testing on day 8. Six independent subjects underwent CO breathing and two muscle biopsies without exercise testing. Molecular studies were performed by real time RT-PCR, Western blot analysis, and immunochemistry. After max testing plus CO breathing, significant increases were found in mRNA levels for nuclear respiratory factor-1 (NRF-1), PGC-1co-activator, mitochondrial transcription factor-A, and DNA polymerase (Pol) with no change in mtDNA copy number or VO₂max. Levels of myosin heavy chain I and nuclear-encoded HO-1, superoxide dismutase-2, citrate synthase, mitofusin-1 and -2 and mitochondrial-encoded cytochrome oxidase subunit-I (COX-I) and ATPase-6 proteins increased significantly. None of these responses were reproduced by VO₂max testing alone, while CO alone increased Tfam and Pol mRNA, and COX-I, ATPase-6, mitofusin-2, HO-1, and SOD2 protein. These findings provide evidence linking the HO/CO response involved in mitochondrial biogenesis in rodents to skeletal muscle in humans through a set of responses involving regulation of the mtDNA transcriptosome and mitochondrial fusion proteins autonomously of changes in exercise capacity.