We studied 134 "healthy persons" with no past history of any serious or latent diseases, and observed differences in their 25 lymphocyte subsets by conducting two-color analyses using 12 useful combinations of dye-labeled monoclonal antibodies. CD4+Leu8+ cells and CD25+CD3+ cells were significantly higher in males than in females, whereas CD4+Leu8- cells and CD23+CD19+ cells were significantly higher in females. In comparison of the 25 lymphocyte subsets among four age groups, CD45RA-CD4+ cells and CDw29+CD4+ cells significantly increased with age, and CD8+ CD11b- cells and CD57-CD8+ cells significantly decreased with age. In females, helper T cells significantly increased and helper inducer T cells increased, while cytotoxic T cells decreased with age. These are important findings that should be considered in studies of immune function and autoimmune disorders.