Cyclosporin A (CsA) is a powerful immunosuppressive drug widely used in transplantation medicine. A major effect of CsA is inhibition of the differentiation of immature double-positive (DP) CD4+ CD8+ thymocytes into mature single-positive (SP) CD4+ CD8- or CD4- CD8+ thymocytes. The mechanisms underlying the changes in CD4/CD8 expression during normal differentiation of thymocytes and the way CsA interferes with this differentiation process are still unknown. Here we show that protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) causes a decrease of both CD4 and CD8 expression at the cell surface level and at the mRNA level in a CD4+ CD8+ T cell line and in freshly isolated thymocytes. A PKC inhibitor, staurosporin, interferes with the differentiation from DP to SP in fetal thymus organ culture system. These data suggest that the alternation of CD4/CD8 expression from DP to SP is dependent on PKC activation. CsA blocks this decrease of CD4/CD8 expression by PMA in vitro. Moreover, this PMA effect is also blocked by treatment with cycloheximide. These results suggest that the reduction of CD4/CD8 expression requires de novo synthesis of a protein(s) induced in response to a signal conveyed by activated PKC. CsA may block the transition from DP to SP by inhibition of CD4/CD8 down-regulation induced by PKC activation.