We have applied the phosphorescence lifetime technique (Vanderkooi, J. M., G. Maniara, T. J. Green, and D. F. Wilson. J. Biol. Chem. 262: 5476-5482, 1987) to determine oxygen tension in single capillaries of the hamster retractor muscle. Palladium meso-tetra(4-carboxyphenyl)porphine (10 mg/ml, pH 7.40, bound to bovine serum albumin) was used as the phosphorescent oxygen sensor. Our measurement system consisted of a microscope configured for epi-illumination, a strobe flash lamp, a 430-nm bandpass excitation filter, and a 630-nm cut-on emission filter. A rectangular diaphragm was used to limit the illumination field to 10 microns x 10 microns, and an end-window photomultiplier tube was used to detect the phosphorescence signal, which was then input to an analog-to-digital board in a personal computer. In vitro calibrations were carried out at 37 degrees C on samples flowing through a glass capillary tube (diameter, 300 microns) at four different O2 concentrations (0, 2.5, 5, and 7.5%). In vivo tests were carried out on arterioles, capillaries, and venules of the retractor muscle of anesthetized hamsters. The phosphorescent compound was administered by injection into a jugular vein (20 mg/kg). Phosphorescence decay curves were analyzed by a new model of heterogeneous oxygen distribution in the excitation/emission volume. Mean Po2 values and the local Po2 gradients within the excitation/ emission volume were calculated from phosphorescence life-times obtained from individual decay curves. The time course of Po2 obtained during 0.5-s measurement periods (5 decay curves at 0.1-s intervals) at a given site along a capillary indicated the presence of a gradient in Po2 within the plasma space between and near red blood cells. Similar Po2 gradients were also detected in arterioles and venules. Mean Po2 values for arterioles, capillaries, and venules over the 0.5-s observation period were 27 +/- 5, 14 +/- 2, and 11 +/- 3 (SD) mmHg, respectively. The magnitude of the Po2 gradient in the arterioles, capillaries, and venules was 6 +/- 1, 4 +/- 1, and 2 +/- 1 mmHg/micron, respectively.