Various isoforms of leukocyte common antigen, or CD45, are expressed differentially on T cells at different stages of development and activation. We report studies on CD45 isoform expression on various subsets of human T cells using two- and three-color flow cytometry and cell depletion. Bone marrow cells that were depleted of CD3+ and HLA-DR+ cells were CD45RARO. The earliest CD3CD4CD8CD19 thymocytes were CD45RO with 20%–30% CD45RA+ cells. The most prominent population of CD4+CD8+ double-positive thymocytes were CD45RARO+. Even the CD4+CD8+ blasts were >90% CD45RO+. About 80% of single-positive thymocytes (CD4+CD8 or CD4CD8+) were also CD45RO+. Only 4.3% of CD4+ and 18% of CD8+ single-positive thymocytes were CD45RA+. In constrast, cord blood T cells which represent the stage that immediately follows single-positive thymocytes, contained 90% CD45RA+ cells. Thus, in terms of CD45 isoform expression, single-positive thymocytes are more like double-positive cells than cord blood T cells. These results suggest the following sequence of CD45 isoform switching during T cell development: CD45RARO or RA+RO (double-negative thymocytes) [RIGHTWARDS ARROW] RARO+ (double-positive and most single-positive thymocytes) [RIGHTWARDS ARROW] RA+RO (cord blood T cells), the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus.