AbstractThe Wiskott‐Aldrich syndrome protein (WASP), which is defective in Wiskott‐Aldrich syndrome (WAS) patients, is an intracellular protein expressed in non‐erythroid hematopoietic cells. Previously, we have established methods to detect intracellular WASP expression in peripheral blood mononuclear cells (PBMNCs) using flow cytometric analysis (FCM‐WASP) and have revealed that WAS patients showed absent or very low level intracellular WASP expression in lymphocytes and monocytes, while a significant amount of WASP was detected in those of normal individuals. We applied these methods for diagnostic screening of WAS patients and WAS carriers, as well as to the evaluation of mixed chimera in WAS patients who had previously undergone hematopoietic stem cell transplantation. During these procedures, we have noticed that lymphocytes from normal control individuals showed dual positive peaks, while their monocytes invariably showed a single sharp WASP‐positive peak. To investigate the basis of the dual positive peaks (WASPlow‐bright and WASPhigh‐bright), we characterized the constituent linage lymphocytes of these two WASP‐positive populations. As a result, we found each WASPlow/high population comprised different linage PBMNCs. Furthermore, we propose that the difference between the two WASP‐positive peaks did not result from any difference in WASP expression in the cells, but rather from a difference in the structural and functional status of the WASP protein in the cells. It has been shown that WASP may exist in two forms; an activated or inactivated form. Thus, the structural and functional WASP status or configuration could be evaluated by flow cytometric analysis.