Blood monocytes as well as tissue-differentiated macrophages play a pivotal role in controlling immune reactions. Monocytes regulate the extent, nature, and duration of immune responses by secretion of cytokines. Interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), IL-10, and IL-12 are of particular interest, since IL-12 shifts the immune response towards a Th1 type, facilitating the production of, e.g., TNF-alpha and IL-6, while IL-10 counteracts Th1 responses and promotes the production of Th2-related cytokines such as IL-4. A tight regulation of these four cytokines keeps the balance and decides whether Th1 or Th2 will predominate in immune reactions. Enzyme-linked immunospot (ELISPOT) assays are among the most-sensitive and -specific methods available for cytokine research. They permit ex vivo identification of individual cells actively secreting cytokines. In the present study we prepared monocytes from healthy subjects' blood and adapted ELISPOT assays to define optimal conditions to detect and enumerate monocytes secreting IL-6, TNF-alpha, IL-10, and IL-12. The optimal time for monocyte incubation was 24 h, and optimal monocyte numbers (in cells per well) were 2,000 for IL-6, 1,000 for TNF-alpha, 50,000 for IL-10, and 100,000 for enumeration of IL-12 secreting monocytes. Among healthy subjects, 10% +/- 5% of the monocytes secreted IL-6, 12% +/- 12% secreted TNF-alpha, 0.1% +/- 0.1% secreted IL-10, and 0.2% +/- 0.3% secreted IL-12 (values are means +/- standard deviations). In conclusion, ELISPOT assays constitute a valuable tool to enumerate monocytes secreting IL-6, TNF-alpha, IL-10, and IL-12 and probably to enumerate monocytes secreting other cytokines and proteins.