Annexin V binding to phosphatidylserine was evaluated by flow cytometry to examine apoptosis in different lymphocyte subsets of peripheral blood mononuclear cells after a 24 h in vitro culture period. We also applied a 2 Gy dose ␥-irradition prior to incubation to evaluate the additional apoptogenic effect of radiation on the lymphocyte subsets. Overall, B lymphocytes showed the highest number of apoptotic cells, followed by T lymphocytes. Within the T lymphocytes, CD4-positive and CD45RAnegative cells were more prone to apoptosis than the CD8-positive and CD45RA-positive cells. Natural killer cells turned out to be most apoptosis-resistant. In the irradiated samples about twice as many apoptotic cells were found and the differences between lymphocyte subpopulations remained. Backgating of the annexin V-positive cells showed that these cells had a clearly decreased forward scatter signal. The antibody binding capacity (ABC) of lymphocyte membrane antigens was determined with CD3fluorescein isothiocyanate (FITC), CD45RA-FITC, CD4-phycoerythrin (PE), CD8-PE, CD56-PE, and CD20-PE in viable and apoptotic cells. In the apoptotic cells a decrease of ABC was found for all antigens, except for CD20. There was no significant cell loss in the cultures. We conclude that the change in scatter and in ABC must be considered in immunophenotyping experiments on cells kept in culture for 24 h. If these changes are taken into account, percentages of subpopulations or the numbers of cells that stain positive for the studied markers do not significantly change. Cytometry 29:242-249, 1997.