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 CD45RA⁻RO⁻. The earliest CD3⁻CD4⁻CD8⁻CD19⁻ thymocytes were CD45RO⁻ with 20%–30% CD45RA⁺ cells. The most prominent population of CD4⁺CD8⁺ double‐positive thymocytes were CD45RA⁻RO⁺. Even the CD4⁺CD8⁺ blasts were >90% CD45RO⁺. About 80% of single‐positive thymocytes (CD4⁺CD8⁻ or CD4⁻CD8⁺) 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: CD45RA⁻RO⁻ or RA⁺RO⁻ (double‐negative thymocytes) → RA⁻RO⁺ (double‐positive and most single‐positive thymocytes) → RA⁺RO⁻ (cord blood T cells), the last switch from CD45RO to CD45RA occurring as a final step of maturation in the thymus.