An investigation of proliferation and activation events in subsets of human CD4+ cells, defined by their expression of CD45RA and CD45R0, is reported. A single-laser based assay for the study of multiple surface antigens and two-parameter cell cycle analysis was used for sorting of and subsequent analysis of proliferation in CD4+CD45RA+ CD45R0-, CD4+CD45RA-CD45R0+ subsets and phenotypically intermediate stages. After labelling with BrdUrd, cells were sorted with flow cytometry on the basis of light-scattering properties and staining with anti-CD45RA, anti-CD45R0, and anti-CD4 markers. Sorted cells were double stained with anti-BrdUrd-antibodies and PI, and the frequencies of proliferating cells were determined. After 48 h, the highest rate of proliferation was found among cells with a phenotype intermediate between CD4+ CD45RA+CD45R0- and CD4+CD45RA-CD45R0+. After 72 h of culture, the situation was changed insofar as the point of highest proliferation had shifted towards the CD4+CD45RA-CD45R0+ population. These findings were further corroborated by four-colour staining with anti-CD4, anti-CD45RA, anti-CD45R0, and Hoechst 33342. This indicates that the phenotype transition is accompanied by cell proliferation. The correlated temporal expression of antigens related to activation (HLA-DR, CD25, CD69, CD71) and cell adhesion (CD11a, CD54, L-selectin) in each of the different subsets was also investigated. All the activation markers CD25, CD69, and CD71 show a more heterogeneous pattern of expression among the CD4+ CD45RA-CD45R0+ cells than the CD4+ CD45RA+CD45R0- cells, indicating a subpopulation of CD4+CD45RA-CD45R0+ cells responding more slowly to the mitogenic stimulation.