Spatial structures of a chymotryptic fragment C2 (residues 1–71) of bacterioopsin from Halobacterium halobium, solubilized in a mixture of methanol/chloroform (1:1, by vol.) and 0.1 M2HCO2NH4, or in perdeuterated sodium (2H)dodecyl sulfate (SDS) micelles in the presence of perdeuterated (2,2,2‐2H)trifluoroethanol, were determined by two‐dimensional and three‐dimensional heteronuclear15N‐1H NMR techniques. The influence of (2,2,2‐2H)trifluoroethanol on the conformational dynamics of C2 in micelles and the effect of the salt (organic mixture) were studied. Under the best conditions,1H and15N resonances of15N‐uniformly enriched protein were assigned in both milieus by homonuclear two‐dimensional NOE (NOESY) and two‐dimensional total‐correlated (TOCSY) spectra and heteronuclear three‐dimensional NOESY‐multiple‐quantum‐correlation (HMQC) and TOCSY‐HMQC spectra. 651 (organic mixture) and 520 (micelles) interproton‐distance constraints, derived from volumes of cross‐peaks in two‐dimensional NOESY and three‐dimensional NOESY‐HMQC spectra, along with deuterium exchange rates of amide groups measured in both milieus and 51 HN‐CαH coupling constants obtained in the case of the organic mixture, were used in the construction of C2 spatial structures. Obtained structures are similar in both milieus and have two right‐handed α‐helical regions stretching from Pro8 to Met32 and Phe42 to Tyr64 (organic mixture), and from Pro8 to Met32 and Ala39 to Leu62 (micelles). In micelles, the second α helix is terminated by C‐cap Gly63, adopting a conformation characteristic of a left‐handed helix. Residues Gly65 to Thr67 form the turn of a right‐handed helix. In the isotropic medium of the organic mixture, the C‐terminal region of residues 65–71 lacks an ordered structure. Torsion angles χ1were unequivocally determined for 18 α‐helical residues in both milieus. In the isotropic organic mixture and anisotropic micellar system, C2 remains a compact structure with a characteristic size of 3.0–3.5 nm. C2 seems to be present in at least two conformational states, packed and unpacked. Using NMR data, along with the electron cryomicroscopy model of bacteriorhodopsin [Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Beckman, E. & Downing, K. H. (1990) J. Mol. Biol. 213, 899–929], we suggested a model for the conformation of C2 in this putative close‐packed state. However, no NOE contact between α helices was found in either milieu. Copyright © 1994, Wiley Blackwell. All rights reserved