We compared the performance of a rapid and simple anti-CD4 antibody-coated microsphere assay with flow cytometry and immunofluorescence for quantitation of absolute count of CD4+ T lymphocytes. A longitudinal evaluation of CD4+ T lymphocytes by flow cytometry and microsphere assay in 10 human immunodeficiency virus (HIV)-seronegative and 59 HIV-seropositive individuals was conducted over a period of 9 months. Standard flow cytometry analysis was performed to establish the absolute CD4+ T-lymphocyte count. The microsphere assay uses whole blood; CD14+ and CD4+ cells are first blocked by small latex beads coated with anti-CD14 antibody, and remaining cells are stained with larger anti-CD4 antibody-coated beads. Cells rosetted with only anti-CD4 antibody-coated beads are counted with use of a hemacytometer. Immunofluorescence microscopy was performed by standard techniques with use of peripheral blood mononuclear cells. The predictive value for stratification of HIV-seropositive patients by CD4+ T-lymphocyte values of < 200/microliters was 95% when the microsphere method was compared with flow cytometry. A correlation coefficient of 0.91 between the two assay methods was demonstrated in 281 CD4+ T-lymphocyte tests for absolute count. Finally, the flow cytometry method yielded better results than did the microsphere assay and immunofluorescence microscopy, in descending order of accuracy. The microsphere method should be effective in determining absolute CD4+ T-lymphocyte count in developing countries where, for a variety of reasons, no other method can be reliably performed.