A new electron paramagnetic resonance (EPRI-based method was developed to obtain selective information on PO, in a specific intracellular compartment (phagosomes). This method did not require the use of a broadening agent thereby eliminating one of the potential sources of experimental error with EPR oximetry. An oxygen-sensitive probe (4-(Tri meth ylammonium) 2,2,6,6-tetramethylpiperidine-d,,-I -oxyl iodide (d-Cat,)) which has a net positive charge, was incorporated selectively into the phagosomes of macrophages stimulated with zymosan. Extracellular oxygen was measured by addition of a neutral nitroxide (4-0x0-2,2,6,6-tetramethylpiperidine-dl6-1 -oxyl (I5N PDT)) to this same sample. Measurements based on EPR linewidths showed the average intraphagosomal oxygen concentration to be 1 1.2 +-3.4 FM lower than that measured from the extracellular compartment when the sample was perfused with air, and this was increased on stimulation of mitochondria1 consumption or by increasing the oxygen concentration in the extracellular compartment. These experiments provide what we believe to be the first reported measurements of the oxygen concentration in a specific intracellular location (intraphagosomal) and its comparison with the oxygen concentration in the extracellular space. The observed gradient cannot be explained in terms of known coefficients of diffusion, and these results are consistent with previous reports that a gradient in oxygen concentration can occur between the average intracellular and extracellular concentration of oxygen.