Cyclosporine A (CsA, 1), an immunosuppressive cyclic undecapeptide, in apolar solvents adopts a II' beta-turn at the Sar3-MeLeu4 residues. [D-Proline3]Cs has been reported to be a nonimmunosuppressive analogue in which the II' beta-turn is retained. In order to determine if this loss of activity is caused by steric hindrance between the Cs analogue and its receptor or is caused by a change in the peptide conformation, an analogue that stabilizes a II' beta-turn has been synthesized, [lactam3,4]Cs. We also have studied the solution conformation of two other analogues, [D-MeAla3]Cs and [L-MeAla3]Cs. The conformations have been established by 1D difference NOE and 2D (NOESY or ROESY) NMR. The conformations of [lactam3,4]Cs and [D-MeAla3]Cs are indistinguishable from that of CsA in solution. [L-MeAla3]Cs was found to adopt a conformation with a cis amide bond between Sar3 and MeLeu4. The inhibition of concanavalin A stimulated thymocytes by CsA, [D-MeAla3]Cs, [L-MeAla3]Cs, and [lactam3,4]Cs gave IC50 values (nM) of 5, 6, 100, and 100, respectively. The weak immunosuppressive activity of [lactam3,4]Cs possessing the II' beta-turn suggests that the loss of activity for 4 is due to steric hindrance with the Cs receptor.