A new EPR-based method was developed to obtain selective information on pO2 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-(trimethylammonium)2,2,6,6-tetramethylpiperidine-d17-1-oxyl iodide (d-cat1)) 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-oxo-2,2,6,6-tetramethyl piperidine-d16-1-oxyl)(15NPDT)) to this same sample. Measurements based on EPR linewidths showed the average intraphagosomal oxygen concentration to be 11.2 q 3.4 fM lower than that measured from the extra-cellular compartment when the sample was perfused with air, and this was increased on stimulation of mitochondrial consumption or by increasing the oxygen concentrations 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. Weare currently involved in experiments to change the composition of the macropha ge membrane so as to influence the observed difference in oxygen concentration.