Since its introduction in 1994 (J. Immunol. Methods 171 (1994) 131), the flow cytometric analysis of lymphocyte proliferation by serial halving of the fluorescence intensity of the vital dye CFSE (carboxyfluorescein diacetate, succinimidyl ester or CFDA-SE) has become widely used in immunological laboratories around the world. This technique allows the visualisation of eight to 10 discrete cycles of cell division by flow cytometry, both in vitro and in vivo. Appropriately conjugated antibodies can be used to probe surface marker changes as cells divide, or changes in expression of internal molecules such as cytokines when appropriate fixation and permeabilisation protocols are used. An added advantage of the technique is the ability to recover viable cells which have undergone defined numbers of cell divisions by flow cytometric sorting, allowing functional studies to be performed. Other commonly used assays of cell proliferation give only limited information, as they usually measure division at a population level. The CFSE technique can be used to determine kinetics of immune responses, track proliferation in minor subsets of cells and follow the acquisition of differentiation markers or internal proteins linked to cell division.