Flow cytometry is a powerful tool in the diagnosis and management of congenital and acquired immunodeficiency syndromes. It is also used for the diagnosis of leukemia and lymphomas. Results of studies from different countries support the presence of variation in absolute and percent quantities of lymphocytes and their subsets (2,3,6,8,12). Several factors were found to affect these results, including age, gender, race, and environmental factors (2, 9, 11). For example, low CD4ϩ -T-cell counts were reported in Asians (1) and Chinese (7). In order to establish a reference range for the different lymphocyte populations, Shahabuddin (8) investigated a cohort of 150 male Saudi blood donors. He showed that there was a marked increase in the CD8 cell count, a decreased CD4/CD8 ratio, and a low natural killer (NK) cell count compared to 32 Caucasian males.In this report, we carried out the immunophenotyping of lymphocytes and their subsets in order to establish our own reference range in an adult Saudi population. MATERIALS AND METHODS Subjects.A total of 209 Saudi men were recruited for this study. These were random blood donors that were used as an internal control each time we conducted an immunophenotyping procedure.Blood collection. EDTA peripheral blood was collected and analyzed initially on Advia 120 (Bayer) hematological analyzer for total and differential blood counts. A blood smear was then prepared for a manual differential count. In brief, 100 l of whole blood was mixed and incubated, in the dark, with 20 l of each monoclonal antibody in separate tubes, at room temperature. Red blood cells were then lysed by adding 2 ml of lysing solution (Becton Dickinson), and the tubes were vortexed and incubated in the dark at room temperature for 10 min and finally centrifuged at 2,500 rpm for 5 min. The pellet was then washed once with 2 ml of phosphate-buffered saline (PBS), resuspended in 500 l of PBS, and finally analyzed with CellQuest software (Becton Dickinson). The FACScalibur was calibrated with Calibrite beads (Becton Dickinson) and AutoComp weekly.Statistical analysis. Data were entered and analyzed by using STATA v6.0 statistical software. The mean and standard deviation (SD) were calculated for each marker. The Student's t test was used to compare the means and SD of our results to those from other published sources.