We have previously identified a developmental sequence among immature thymocytes, prior to their expression of the lineage markers CD3, CD4, and CD8. This sequence is marked by transient expression of the interleukin 2 receptor alpha chain (IL 2R alpha). The most mature cells in this sequence (surface phenotype heat-stable antigen (HSA)++ Pgp-1- IL 2R alpha-) are the immediate precursors to CD4+CD8+ small cortical thymocytes, and have by definition been considered to be CD4-CD8-. We now show that these cells display low levels of surface CD4 and CD8, but not CD3. This low-level expression begins to appear immediately after the loss of IL 2R alpha expression. Northern blot analysis for mRNA expression confirms that these IL 2R alpha- cells are transcribing CD4 and CD8 mRNA, in contrast to their immediate (IL 2R alpha+) precursor. Upon unstimulated culture, these IL 2R alpha- cells gradually acquire high levels of CD4 and CD8, as well as low levels of CD3, whereas IL 2R alpha+ cells do not. These findings suggest that the IL 2R alpha+ subset is the end of the true CD3-CD4-CD8- phase, and that the intracellular signals for CD3, CD4, and CD8 acquisition occur simultaneously with, or immediately prior to, the signal for down-regulation of IL 2R alpha.