A method to detect single tumor necrosis factor alpha (TNF alpha)-producing cells, based on immunofluorescence staining, is presented. We have developed a rapid and simple procedure to permeabilize blood leukocytes for antibodies without causing cell aggregation. Using monoclonal as well as polyclonal TNF alpha-specific antibodies cytoplasmic TNF alpha could be detected in lipopolysaccharide (LPS)-stimulated monocytes in identical ways. In addition, one out of 15 tested fresh blood samples from healthy blood donors contained occasional TNF alpha-producing cells. We found a well-defined staining of the intracellular TNF alpha with a local, polar accumulation in a juxtanuclear position of the cell. This finding most probably indicated the presence of the monokine in the Golgi organelle because the sequential staining for TNF alpha and the Golgi zone by specific antibodies coincided. Thus, we believe the TNF alpha accumulation at this site reflects the production rather than cellular uptake of the cytokine, since the incubation of blood leukocytes with recombinant TNF alpha did not lead to any detectable staining. By performing two-color staining of cell surface antigens and cytoplasmic TNF alpha of LPS-stimulated blood leukocytes we could demonstrate that monocytes exclusively contributed to the TNF alpha synthesis. At the peak of the response, which occurred 2-3 h after LPS exposure, 50-60% of the blood monocytes produced TNF alpha. We noted a rapid decline in the number of TNF alpha-producing cells already 6 h after initiation of these cultures.