Phosphorescence quenching techniques measure microvascular PO2 without direct surgical manipulation of the tissue. At a given arterial PO2, microvascular PO2 reflects the local O2 uptake-to-O2 delivery ratio, i.e., VO2/QO2. We evaluated the potential of phosphorescence quenching to determine microvascular PO2 in the rat costal diaphragm (PdiaO2). PdiaO2 and arterial blood gases were monitored across transient changes of inspired O2 among 21, 10, and 100% and also during hypotensive states evoked by progressive phlebotomy. After a transit delay, PdiaO2 responded rapidly to alterations of inspired and thus arterial PO2, with half times of the response averaging 5-7 s for switches to a lower inspired O2 (i.e., from 21 to 10%, from 100 to 21%, and from 100 to 10%) and also from 10 to 21%. By comparison, half times of the 10 to 100% and 21 to 100% switches were longer [11 s (P = 0.085) and 21 s (P < 0.05), respectively]. Below a mean arterial blood pressure (BP) of 120-130 mmHg, PdiaO2 decreased as a linear function of BP, with these variables being significantly correlated in each instance (n = 5, r = 0.851-0.937, P < 0.01 for all animals). From these results, it appears feasible to measure PdiaO2 in the spontaneously breathing rat in vivo under steady-state and transient conditions. Also, during progressive hypotension, the fall in PdiaO2 is significantly related to falling BP, likely as a consequence of an increased metabolic demand (increased ventilation and diaphragm VO2) concomitant with decreased blood flow. We conclude that phosphorescence quenching techniques offer a powerful tool for assessing diaphragm physiology and pathophysiology.