Development of a more cost-effective and efficient method of performing lymphocyte subset analysis is of continuing importance in clinical flow cytometry laboratories. Current two-color methods utilize forward and right angle light scatter and multiple tubes per sample and are thereby liable to gate contamination. Methods using three-color analysis with CD45 vs. right angle light scatter ( R A N gating cannot always exclude a contaminating nonlymphoid population. We have established a two tube approach to directly measure total T cells, T suppressor, and T helper subsets, total B cells and total natural killer cells. The technique involves staining of whole blood with a mixture of five monoclonal antibodies conjugated to three fluorochromes: CD4+ CD19 fluorescein isothiocyanate (FITC), CD3 + CD33 phycoerythrin (PE), CD45 peridin chlorophyll alpha protein (PerCP), CD8+CD16 FITC, CD3+CD33 PE, and CD45 PerCP. Analysis is performed using a single laser flow cytometer. This method has equivalent recovery to and improved purity of the lymphocyte gate when compared to well-established methods. These antibody combinations additionally allow clear separation of lymphocytes from other leukocytes and debris as well as separation of the T cell helper and suppressor subsets, natural killer cells and B lymphocytes. We additionally provide preliminary data that an accurate lymphocyte subset analysis can be performed on a single tube containing five antibodies (CD4+ CD19 FITC, CD3 + CD33 PE, and CD45 PerCP), although some measurements are performed deductively. @ 1996 Wiley-Liss. Inc.