The assumption that cellular oxygen pressure (PO2) is close to zero in maximally exercising muscle is essential for the hypothesis that O2 transport between blood and mitochondria has a finite conductance that determines maximum O2 consumption. The unique combination of isolated human quadriceps exercise, direct measures of arterial, femoral venous PO2, and 1H nuclear magnetic resonance spectroscopy to detect myoglobin desaturation enabled this assumption to be tested in six trained men while breathing room air (normoxic, N) and 12% O2 (hypoxic, H). Within 20 s of exercise onset partial myoglobin desaturation was evident even at 50% of maximum O2 consumption, was significantly greater in H than N, and was then constant at an average of 51 +/- 3% (N) and 60 +/- 3% (H) throughout the incremental exercise protocol to maximum work rate. Assuming a myoglobin PO2 where 50% of myoglobin binding sites are bound with O2 of 3.2 mmHg, myoglobin-associated PO2 averaged 3.1 +/- .3 (N) and 2.1 +/- .2 mmHg (H). At maximal exercise, measurements of arterial PO2 (115 +/- 4 [N] and 46 +/- 1 mmHg [H]) and femoral venous PO2 (22 +/- 1.6 [N] and 17 +/- 1.3 mmHg [H]) resulted in calculated mean capillary PO2 values of 38 +/- 2 (N) and 30 +/- 2 mmHg(H). Thus, for the first time, large differences in PO2 between blood and intracellular tissue have been demonstrated in intact normal human muscle and are found over a wide range of exercise intensities. These data are consistent with an O2 diffusion limitation across the 1-5-microns path-length from red cell to the sarcolemma that plays a role in determining maximal muscle O2 uptake in normal humans.