Many clinical situations demand repeated analyses of blood parameters but permit only minimal amounts of peripheral blood to be taken, e.g., in neonates with low birth weight, during extensive operations of young children, or in patients with restricted bone marrow function. In these cases laser scanning cytometry is the ideal tool to determine the distribution of different leukocyte-subsets. The purpose of this protocol is to describe stepwise a new method of immunophenotyping by laser scanning cytometry. In this assay nuclear DNA is stained by 7-aminoactinomycin-D (7-AAD) and surface antigens are detected by direct three-colour immunofluorescence. For data acquisition, measurements are triggered on the 7-AAD-fluorescence. Data are obtained for forward scatter, green, orange, and long red fluorescence by excitation with the argon-laser, and for far red fluorescence by excitation with the helium-neon-laser. Using this protocol the amount of peripheral blood needed is minimised to 10 microl. Specimens can be stained a second time in a different way and analysed repeatedly and archived.