Most thymocytes are either immature or functionally deficient and express a series of lymphocyte cell-surface antigen markers designated Lyt 1, Lyt 2 and Lyt 3 (refs 1, 2) which have been useful in distinguishing functional subsets of T cells. In contrast, a small population of cortisone-resistant thymocytes (CRT), confined to the thymic medulla after acute corticosteroid treatment are functionally more mature. These cells, like peripheral T cells, have restricted expression of Lyt antigens and mostly are either Lyt 1 or Lyt 123 cells. It has thus been assumed that all thymocytes initially are Lyt 1+, 2+, 3+ and by differentiation lose either Lyt 1 or Lyt 2, 3 to result in Lyt 1+(23-) and Lyt (1-)23+ cells. Using immunofluorescence (IF) and flow microfluorometry (FMF) analyses to detect Lyt antigen expression quantitatively without the requirement for cell lysis, we have now re-examined the expression of Lyt 1, 2 and 3 antigens on murine fetal thymocytes from 14 to 19 days of gestation and on normal thymocytes from birth to 2-3-month-old adults. These studies demonstrate that cells with the Lyt 1+23- phenotype first appear in the thymus several days before Lyt 123+ thymocytes are detected, and suggest either a micro-environmental or site-specific influence for phenotypic differentiation and/or two independent, pre-committed lineages.