Cyclosporine mediates its immunosuppressive effect by preventing the synthesis of lymphokine mRNA during the process of T lymphocyte activation. Although the detailed molecular mechanism by which CsA achieves this effect is unknown, two proteins have been identified as putative intracellular CsA-receptor proteins. One of these, calmodulin, is an important Ca++-binding protein and enzyme cofactor and the other, cyclophilin, is a novel protein that is reported to have protein kinase activity. In this study the CsA-binding capacity of both these proteins has been assessed using CsA-coated ELISA plates and CsA-affinity gel matrices. CsA binding was shown by cyclophilin whereas no CsA-calmodulin binding could be detected under identical conditions. However, it was not possible to demonstrate any cyclophilin-associated protein kinase activity. Jurkat cells were probed for the presence of CsA-binding proteins using the CsA-affinity gel matrix; a 17 KD protein, most probably cyclophilin, was identified as the major CsA-binding protein. In addition, a previously unidentified CsA-binding 45 KD phosphoprotein was precipitated from 32P-labeled Jurkat cells. These results would support cyclophilin as the major, if not only, intracellular receptor protein for CsA. However, the relationship between binding of CsA to cyclophilin and/or the 45 KD phosphoprotein and the immunosuppressive effects of CsA is still unknown.