Thymocytes can be separated into 5 subsets defined by their surface expression of CD4, CD8, and CD5. These 5 subsets include: 1) the double negatives (DN; CD4-, CD8-, dull and bright CD5+) which contain the most immature cells, 2) the dull Ly-1 (dLyl, CD4-, CD8-, dull CD5+) a subset of DN, 3) double positives (DP; CD4+, CD8+, CD5+) an intermediate stage of differentiation, 4) CD4+ (CD4+, CD8+, CD5+) a mature subset, and 5) CD8+ (CD4-, CD8+, CD5+) another mature subset. This project focuses on the phenotype, differentiation and regulation of these thymocyte subsets. The phenotype of rat and louse DN thymocytes has been compared. The majority of both rat and mouse DN cells are proliferating when first isolated. However in remarkable contrast to the mouse, the rat DN thymocytes reveal no interleukin 2 receptor (IL-2R) expression. They are also absent of detectable IL-2 mRNA and cytoplasmic IL-2 activity. Examination of estradiol resistant dLyl thymocytes has shown that the dLyl cells remaining in the thymus after short-term estradiol treatment are depleted of bright IL-2R+, large and blast cells yet are still able to generate all CD4 and/or CD8 thymic subsets in irradiated recipients. We have compared the anatomic localization of donor-derived thymocytes following the transfer of various thymic precursors into irradiated recipients. It was found that donor-derived thymocytes are first seen at day 12, after bone marrow cell transfer and are located in the medulla. In contrast, after dLyl transfer, donor-derived cells are first detected by day 6 and are in a speckled pattern throughout all regions of the thymus. Studies on the expression of ets oncogenes in thymocyte subsets has demonstrated that in the adult thymus, ets-1 and ets-2 MRNA expression is 8- to 10-fold higher in the CD4+ subset than in the other subsets-examined. Moreover, both the CD4+ and the CD8+ T cell subsets had lower ets RNA levels than the CD4+ thymocytes. Finally, we have observed that the DNA isolated from either CD4+ or CD8+ thymocytes, the more mature thymic subsets, is less methylated in the TCR- beta region than DNA isolated from the DN population containing the more immature thymocytes.