Orientational constraints generated from solid-state NMR of uniformly aligned gramicidin A in hydrated lipid bilayers have been used to determine the indole ring orientations for the four tryptophans of the gramicidin A monomer with respect to the bilayer normal and the channel axis. 15N epsilon 1 labeled tryptophan has been incorporated into gramicidin at positions 9, 11, 13, and 15. The chemical shift tensor orientation has been oriented with respect to the N-H bond via doubly labeled sample in which the 15N epsilon 1-1H has been exchanged for 2H. By observation of the dipolar coupled 15N chemical shift powder pattern of the amino acid, sigma cc has been shown to be perpendicular to the plane of the ring and that sigma aa makes an angle of 25 degrees with respect to the N-H bond. The indole ring orientations were obtained from a consideration of both the chemical shift and the 15N-1H dipolar interaction. These four rings have very similar orientations with respect to the bilayer normal as given by the range of angles between the bilayer and ring normals (64-67 degrees). Furthermore, the N-H bond orientations with respect to the bilayer normal varies by only 10 degrees among the four sites. This orientational analysis has been based on an assumption that large amplitude librational motions in the hydrated bilayer samples are not averaging the nuclear spin interactions. This assumption was verified by analyzing the 2H quadrupole spectra of d5-Trp11-labeled gramicidin A in oriented preparations. The orientations predicted for the five C-H bonds in the indole ring from the 15N data agreed (root-mean-square deviation of 3.7 degrees) with the observed orientations from quadrupole splittings of the C-D bonds in the ring. From the orientation of the indole rings with respect to the bilayer normal and a polypeptide backbone conformation, the four typtophans of the gramicidin monomer are oriented with respect to the backbone of the channel conformation. The similarity among the indole orientations with respect to the bilayer normal is therefore consistent with the electrophysiological results that the individual replacement of the indole rings with phenyl rings results in a incremental decrease in the conductance of the channels formed. The indole orientations with respect to the backbone as defined by the side-chain torsion angles is not uniquely determined but yields a discrete set of possible values.(ABSTRACT TRUNCATED AT 400 WORDS)