We have used experimental deuterium NMR spectra from labeled tryptophans in membrane-spanning gramicidin A (gA)(1) channels to refine the geometry of the indole ring and, specifically, the C2-H-2 bond direction. By using partial exchange in a cold organic acid, we were able to selectively deuterate ring positions C2 and C5 and, thereby, define unambiguous spectral assignments. In a backbone-in dependent analysis, the assigned spectra from four distinct labeled tryptophans were used to assess the geometry of the planar indole ring. We found that the C2-H-2 bond makes an angle of about 6degrees with respect to the normal to the indole ring bridge, and the experimental geometry was confirmed by density functional calculations using a 6-311G** basis set. The precisely determined ring geometry and the experimental spectra in turn are the foundation for calculations of the orientation of each tryptophan indole ring, with respect to the bilayer membrane normal, and of a principal order parameter S-zz for each ring. The results have general significance for revising the tryptophan ring geometry that is used in protein molecular modeling, as well as for the analysis of tryptophan ring orientations in membrane-spanning proteins. The experimental precision in the definition of the indole ring geometry demonstrates yet another practical application emanating from fundamental research on the robust gramicidin channel.