Decrease of ambient oxygen level has been used in myocytes culture experiments in examining the responsiveness to stress secondary to hypoxia. However, none of these studies measure the myocytes oxygenation levels resulting in ambiguity as to whether there is insufficient oxygen delivery. This study examined the hypothesis that at a basal myocardial work state, adequate myocyte oxygenation would be maintained until extremely low arterial pO2 levels were reached. Myocyte pO2 values in normal dogs were calculated from the myocardial deoxymyoglobin (Mb- λ) levels using 1H-spectroscopy (MRS) and were normalized to Mb-λ obtained after complete LAD occlusion. During Protocol 1 (n = 6), Mb-λ was measured during sequential reductions of the oxygen fraction of inspired gas (FIO2) from 40, 21, 15, 10, and 5%, while in protocol 2 (n = 10) Mb-λ was measured at FIO2 of 3%. Protocol 3 (n = 9) evaluated time course of Mb-d during prolonged exposure to FIO2 of 5%. Myocardial blood flow (MBF) was measured with microspheres and high energy phosphate (HEP) levels were determined with 31P-MRS. MVO2 progressively increased in response to the progressive reduction of FIO2 that is accompanied by increased LV pressure, heart rate, and MBF. Mb-λ was undetectable during FIO2 values of 21, 15, 10, and 5%. However, FIO2 of 3% or prolonged exposure to FIO2 of 5% caused progressive increases of Mb-d which were associated with decreases of PCr, ATP and the PCr/ATP ratio, as well as increases of inorganic phosphate. The intracellular PO2 values for 20% reductions of PCr and ATP were approximately 7.4 and 1.9 mmHg, respectively. These data demonstrate that in the in vivo system over a wide range of FIO2 and arterial pO2 levels, the myocyte pO2 values remain well above the Km value with respect to cytochrome oxidase, and oxygen availability does not limit mitochondrial oxidative phosphorylation at 5% FIO2.