Abstract: Binary complex formation between the immunosuppressive drug cyclosporin A (CsA) and cyclophilin 18 is the prerequisite for the ability of CsA to inhibit the protein phosphatase activity of calcineurin, a central mediator of antigen-receptor signaling. We show here that several CsA derivatives substituted in position 3 can inhibit calcineurin without prior formation of a complex with cyclophilin 18. [Methylsarcosine(3)]CsA was shown to inhibit calcineurin, either in its free form with an IC(50) value of 10 microm, or in its complex form with cyclophilin 18 with an IC(50) of 500 nm. [Dimethylaminoethylthiosarcosine(3)]CsA ([Dat-Sar(3)]CsA) was found to inhibit calcineurin on its own, with an IC(50) value of 1.0 microm, but was not able to inhibit calcineurin after forming the [Dat-Sar(3)]CsA-cyclophilin 18 binary complex. Despite their different inhibitory properties, both CsA and [Dat-Sar(3)]CsA suppressed T cell proliferation and cytokine production mainly through blocking NFAT activation and interleukin-2 gene expression. Furthermore, to demonstrate that [Dat-Sar(3)]CsA can inhibit calcineurin in a cyclophilin-independent manner in vivo, we tested its effect in a Saccharomyces cerevisiae strain (Delta12), in which all the 12 cyclophilins and FKBPs were deleted. [Dat-Sar(3)]CsA, but not CsA, bypassed the requirement for cellular cyclophilins and caused growth inhibition in the salt-stressed Delta12 strain.