Cyclosporin A (CsA) inhibits the development of mature thymocytes from their CD4+ CD8+ precursors, but may allow autoreactive cells to mature. Using 3-color flow cytometry, we have followed the progressive development of thymocytes, including potentially autoreactive cells, during CsA treatment. Numbers of CD4+ CD8+ CD3high thymocytes dropped immediately, suggesting that the generation of these mature thymocyte precursors, normally dependent upon positive selection, was inhibited by CsA. Numbers of CD4+ CD8- thymocytes also declined rapidly, but CD4 - CD8+ thymocytes were unaffected for 2 days, suggesting that the mature single-positive subsets are not symmetrically derived from a common GsA-sensitive precursor. An exceptional subset of CD8 SP thymocytes, expressing CD45RA, did not respond to CsA for about 10 days, indicating that they are distantly derived from a CsA-sensitive precursor. Apoptosis of TCR-V beta 3 + thymocytes caused by Mtv-6, quantified according to the down-regulation of CD4 and CD8 on immature thymocytes, was partially inhibited by CsA, to maximal effect within 24 hours. This did not, however, facilitate their development into mature thymocytes.