To dissect the enzyme inhibitory properties of the immunosuppressive cyclic undecapeptide cyclosporin A (CsA) and gain access to monospecific, non-calcineurin-inhibiting CsA derivatives, [D-Ser8]CsA was subjected to modifications at the D-Ser side chain. Thus, we modified a CsA residue flanking the calcineurin (CaN) and cyclophilin 18 (Cyp18) binding domains of CsA instead of the residues of the CaN binding domain in order to develop a new specificity-determining site within the cyclic peptide. The [O-(NH2 (CH2)5NHC(O)CH2)-D-Ser8]CsA (derivative 9), with an amino group on a tether, exhibits CsA-like inhibition of the peptidyl prolyl cis/trans isomerase activity of Cyp18 with an IC50 value of 3.2 nm, whereas the CaN inhibition by the Cyp18-derivative 9 complex is completely abolished. Consequently, this compound is not able to inhibit the proliferation and cytokine production of activated T cells. Structure-activity relationship studies with a series of [d-Ser(8)]CsA derivatives indicate that the positively charged side chain is an essential requirement for Cyp18-derivative 9 to be ineffective on CaN. Upon protecting the amino group in derivative 9 with the photolabile moiety 2-nitroveratryloxycarbonyl (NVOC), the Cyp18-[O-(NVOC-NH(CH2)5NHC(O)CH2)-D-Ser8]CsA (derivative 11) complex exhibits strong CaN inhibition and shows potent immunosuppressive activity. In stimulated T cells pretreated with derivative 11, a remarkable recovery of transcriptional activation of the nuclear factor of activated T cells (NFAT) has been achieved through light irradiation, as assessed with a NFAT reporter gene assay.