Analysis of the functional activity of phagocytes is of great importance in the differential diagnosis of patients with recurrent bacterial infections. Here we describe a method to determine the production of reactive oxygen intermediates (ROI) by microcytofluorometry using dihydrorhodamine 123, a derivative of rhodamine 123. Using this method the ROI production of erythrocyte-depleted whole blood samples can be measured without further time-consuming purification steps. Possible harmful manipulation of the isolated cells can also be avoided and highly reproducible and significant results are obtained in the minimum of time. This assay provides a very sensitive alternative to the clinically used NBT test in the diagnosis of patients with chronic granulomatous disease (CGD). Moreover, the analysis of oxygen-dependent effector functions of murine effector cells and cell lines may be important in investigating resistance to certain microbes (e.g., Candida albicans, Staphylococcus aureus or different protozoa such as Toxoplasma gondii or Leishmania species).