Background: Absolute counts of CD4(+) T-lymphocytes are used in the management of patients with human immunodeficiency virus infection. Low absolute counts of CD3(+)CD4(+) cells have also been observed in healthy people-a phenomenon called idiopathic CD4 lymphocytopenia. It is common practice for normal ranges for lymphocyte subsets to be derived from samples taken from blood donors. Study Design and Methods: A sample of EDTA blood was taken through the donation line tubing, after donation from 565 blood donors in Sydney, Australia, who were selected from a range of age groups. An additional 12 donors provided a predonation sample as well as a postdonation sample. Hematologic assays were performed on two analyzers, Samples were stained for CD3, CD4, CD8, CD19, and CD56 and analyzed on a flow cytometer. Results: Three donors were found to have absolute CD3(+)CD4(+) counts <300 cells per mu L. The percentage of CD3(+)CD4(+) cells was found to increase with age. Both the percentage and the absolute count of CD3(+)CD8(+) cells decreased with age, which resulted in an increased CD4:CD8 ratio with age. Men had consistently higher absolute counts of CD3-CD56(+) cells than women. The 12 additional donors all had greater percentages of CD3(+)CD4(+) cells and lower absolute counts for CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD19(+) and CD3(-)CD56(+) cells after donation than they had before donation (p<0.001). Conclusion: It is not satisfactory to base normal ranges for lymphocyte subsets on donor blood, from which the blood sample has been obtained after donation.