Cyclophilin (163 residues, Mτ 17737), a peptidyl prolyl cis-trans isomerase, is a cytosolic protein that specifically binds the potent immunosuppressant cyclosporin A (CsA). The native form of the major bovine thymus isoform has been analyzed by 2D NMR methods, COSY, HOHAHA, and NOESY, in aqueous media. The 156 main-chain amides in CyP yield 126 observable NH/αCH couplings (81%, Gly pairs counted as 1). Following exhaustive D2O exchange, 44 amide resonances remain visible. Further analysis of the NH/NH, NH/αCH, and αCH/αCH regions of the COSY and NOESY data sets indicates that the residual amides in D2O form a coherent hydrophobic domain which yields 2D NMR features suggestive of a β-sheet. Many (43/126) of the amide resonances have been classified according to amino acid type. In the aromatic region of the spectra, the assignment of the ring spin systems is nearly complete (12/15 Phe, 2/2 Tyr, 1/1 Trp, and 3/4 His). This has successfully lead to the complete assignment of all of their βCH’s, main-chain αCH resonances, and many of the backbone amide resonances (8/12 Phe, 2/2 Tyr, 1/1 Trp, and 2/3 His). In other regions of the spectrum, the side-chain and main-chain resonances for 10/23 Gly, 9/9 Ala, 5/11 Thr, 5/9 Val, and 1/6 Leu have been completely assigned. The drug-free cyclophilin and CsA-bound cyclophilin form two discrete protein structures that are in slow exchange on the NMR time scale. Comparison of the fingerprint regions from the COSY spectra obtained from the two forms of the protein reveals a minimum of 16 cross-peaks which are clearly shifted upon complexation. In fact, on the basis of chemical shift changes observed in assigned side-chain and main-chain resonances, only a relatively few of the amino acid residues identified to date are perturbed by complex formation. These include 3 Phe (8, 12, and 14) and the Trp in the aromatic region and 2 Ala (7 and 8) in the Ala/Thr region. In the upfield-shifted methyl region, an assigned Leu and Val spin system and a spin system labeled X10 (an Ile or Leu) are affected by complex formation. In addition, a new aliphatic spin system, labeled XII, which shows a close spatial relationship to the perturbed Phe 12, is observed in this region of the spectrum. In summary, the regions of the protein altered by complex formation can be divided into two categories: a hydrophobic and a H2O-accessible domain. The hydrophobic region, defined in the D2O exchange data sets, involves seven of the sixteen shifted NH/αCH COSY cross-peaks and includes Leul, Vall, and Phel2. The other nine perturbed NH/αCH COSY cross-peaks occur in a region of the protein that is accessible to D2O exchange in drug-free cyclophilin and includes the Trp, Ala7, Ala8, and X10 residues.