BACKGROUND: In many cases a frequent monitoring of blood is important, e.g. during an infection. Often the availability of blood is the limiting factor. METHODS: 50 microl blood were stained and analyzed using a standard four-color cytometer. Percentages of leukocytes were calculated by FlowJo software. RESULTS: Our protocol allows the differentiation of B cells, T cells, NK cells, neutrophils, and monocytes/macrophages in small volumes of blood. CONCLUSIONS: Using nine fluorochrome-labeled antibodies and a specific gating strategy we were able to differentiate the main immune cells in minute amounts using one staining step.