We have used PKH26 dye, which is incorporated stably into the membrane of cells, to determine, using flow cytometry, lymphocyte proliferative responses to the antigen tetanus toxoid in fresh and cryopreserved samples. Measuring cell proliferation with this dye has advantages over either 3H-thymidine or Bromodeoxyuridine (BrdU). Whereas the existing methods measure proliferation at a single time point, PKH26 gives a cumulative measure of cell proliferation. As PKH26 is incorporated into the cell membrane, cells do not have to be permeabilised to allow dye incorporation into a cytoplasmic compartment. Most importantly, PKH26 can be used in combination with monoclonal antibodies to surface markers on mixed populations of cells, to determine the proliferation of individual subpopulations, without the need for prior cell fractionation. We also show that PKH26 can be used with similar efficacy in both fresh and cryopreserved samples. In addition since PKH26 is a cumulative measure of proliferative responses we were able to show that restimulation of the dividing population in vitro with fresh antigen presenting cells (APC) and antigen permits characterisation of a further proliferating cell population. The use of PKH26 dye in combination with cell phenotyping and measurement of cytokine production at the single cell level will prove a powerful tool for multiparameter analyses of cellular responses to antigen.