The effects of cyclosporin A (CsA) on T-cell development were assessed in an organ culture of murine foetal thymus. Applying three-colour flow cytometric analysis, we showed that the agent inhibits the development of mature CD3/T-cell receptor alpha beta (TcR alpha beta)+ cells both in CD4+8- and CD4-8+ populations. CD4-8- cells appeared to be accumulated by CsA. We examined the heterogeneity of CD4-8- cells generated in the organ culture, and defined five subpopulations by the expression of the cell-surface molecules CD3/TcR, J11d and CD25. It has been demonstrated that only the CD3/TcR alpha beta+ J11d- CD25- subpopulation is susceptible to the suppressive effects of CsA among CD4-8- cells, whereas all the other four subpopulations, including CD3/TcR gamma delta+ cells, are resistant. Thus, all of the TcR alpha beta-bearing cells, including CD4-8- cells but none of the TcR alpha beta- cells, are CsA sensitive. Because it is known that CsA inhibits the TcR-mediated signalling events in mature T cells and that signallings mediated via the interaction of TcR with major histocompatibility complex (MHC) molecules on thymic stroma cells are crucial for thymic selection of T cells, these results indicate that TcR alpha beta-bearing CD4-8- cells but not TcR gamma delta-bearing CD4-8- cells undergo thymic positive selection.