Mitochondria utilize the majority of oxygen (O₂) consumed by aerobic organisms as the final electron acceptor for oxidative phosphorylation (OXPHOS) but also to generate reactive oxygen species (mtROS) that participate in cell signaling, physiological hormesis, and disease pathogenesis. Simultaneous monitoring of mtROS production and oxygen consumption (Jo₂) from tissue mitochondrial preparations is an attractive investigative approach, but it introduces dynamic changes in media O₂ concentration ([O₂]) that can confound experimental results and interpretation. We utilized high-resolution fluorespirometry to evaluate Jo₂ and hydrogen peroxide release (Jh₂o₂) from isolated mitochondria (Mt), permeabilized fibers (Pf), and tissue homogenates (Hm) prepared from murine heart and skeletal muscle across a range of experimental [O₂]s typically encountered during respirometry protocols (400-50 µM). Results demonstrate notable variations in Jh₂o₂ across tissues and sample preparations during nonphosphorylating (LEAK) and OXPHOS-linked respiration states at 250 µM [O₂] but a linear decline in Jh₂o₂ of 5-15% per 50-µM decrease in chamber [O₂] in all samples. Jo₂ was generally stable in Mt and Hm across [O₂]s above 50 µM but tended to decline below 250 µM in Pf, leading to wide variations in assayed rates of Jh₂o₂/O₂ across chamber [O₂]s and sample preparations. Development of chemical background fluorescence from the H₂O₂ probe (Amplex Red) was also O₂ sensitive, emphasizing relevant calibration considerations. This study highlights the importance of monitoring and reporting the chamber [O₂] at which Jo₂ and Jh₂o₂ are recorded during fluorespirometry experiments and provides a basis for selecting sample preparations for studies addressing the role of mtROS in physiology and disease.

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