A whole-blood flow cytometry-based assay was utilized to assess CD4 and CD8 T-lymphocyte activation in response to phytohemagglutinin (PHA) stimulation. T-lymphocyte activation was assessed by qualitative (percent CD69) and semiquantitative (anti-CD69 antibody binding capacity) measurements of CD69 surface expression. Whole-blood samples from 21 healthy and 21 human immunodeficiency virus (HIV)-infected (<500 absolute CD4 counts per mm³) individuals were stimulated with 20 µg of PHA per ml for 18 to 24 h. The proportions of activated CD4 and CD8 T lymphocytes expressing CD69 (percent CD69) and the levels of CD69 expression on each T-lymphocyte subset (anti-CD69 antibody binding capacity) were measured. By using this assay system, T-lymphocyte activation was impaired in both CD4 and CD8 T-lymphocyte subsets of HIV-infected individuals. The proportions of CD69-positive CD4 and CD8 T lymphocytes were 43 and 27% lower, respectively, in samples from HIV-infected individuals compared to samples from healthy individuals. Similarly, the levels of CD69 expression on each activated CD4 and CD8 T-lymphocyte subset were 48 and 51% lower, respectively. These results suggest that both qualitative and semiquantitative measurements of CD69 surface expression by flow cytometry can be used to assess T-lymphocyte activation.