There exists substantial controversy as to whether muscle oxygen (O2) delivery (QO2) or muscle mitochondrial O2 demand determines the profile of pulmonary VO2 kinetics in the rest-exercise transition. To address this issue, we adapted intravascular phosphorescence quenching techniques for measurement of rat spinotrapezius microvascular O2 pressure (PO2m). The spinotrapezius muscle intravital microscopy preparation is used extensively for investigation of muscle microcirculatory control. The phosphor palladium-meso-tetra(4-carboxyphenyl)porphyrin dendrimer (R2) at 15 mg/kg was bound to albumin within the blood of female Sprague-Dawley rats ( approximately 250 g). Spinotrapezius blood flow (radioactive microspheres) and PO2m profiles were determined in situ across the transition from rest to 1 Hz twitch contractions. Stimulation increased muscle blood flow by 240% from 16.6 +/- 3.0 to 56.2 +/- 8.3 (SE) ml/min per 100 g (P < 0.05). Muscle contractions reduced PO2m from a baseline of 31.4 +/- 1.6 to a steady-state value of 21.0 +/- 1.7 mmHg (n = 24, P < 0.01). The response profile of PO2m was well fit by a time delay of 19.2+/-2.8 sec (P < 0.05) followed by a monoexponential decline (time constant, 21.7 +/- 2.1 sec) to its steady state level. The absence of either an immediate and precipitous fall in microvascular PO2 at exercise onset or any PO2m undershoot prior to achievement of steady-state values, provides compelling evidence that O(2) delivery is not limiting under these conditions.