Three analogues of the helical ionophore gramicidin A have been synthesized with (13)C-labeled carbonyls ((13)C=O) incorporated at either Gly(2), Ala(3), or Val(7). A fourth compound incorporated (13)C at both the carbonyl and alpha-carbon of Gly(2) within the same molecule. These labels were studied using solid-state, proton-enhanced, (13)C nuclear magnetic resonance (NMR) in hydrated dispersions of dimyristoylphosphatidylcholine (DMPC)-gramicidin A. The dispersions were aligned on glass coverslips whose orientation to the magnetic field could be varied through 180 degrees . The orientation dependence of the NMR spectrum was used to obtain an accurate measurement of the (13)C chemical shift anisotropy (CSA), and in the case of the fourth compound, the (13)C-(13)C dipolar coupling constant. From the measured CSA and estimates of the orientation of the (13)C shielding tensor, we are able to determine the direction of the (13)C=O bonds and to compare these with the predictions of the various reported models for the configuration of gramicidin A in phospholipid bilayers. Our results are consistent with the left-handed pipi(6.3) (LD) single-stranded helix (Urry, D. W., J. T. Walker, and T. L. Trapane. 1982. J. Membr. Biol. 69:225-231). The right-handed pipi(6.3) (LD) single-stranded helix observed for gramicidin A in sodium dodecyl sulfate micelles (Arseniev, A. S., I. L. Barsukov, V. F. Bystrov, A. L. Loize, and Yu A. Ovchinnikov. 1985. FEBS (Fed. Eur. Biochem. Soc.) Lett. 186:168-174) yields a poorer fit to the data. However, the width of the carbonyl resonances suggests a distribution of molecular geometries possibly resulting from a spread in the helix pitch and handedness. Double-stranded helices and beta sheet structures are excluded. In dispersions in which the lipid is in the L(alpha) phase, the gramicidin A undergoes rapid reorientation about an axis which is centered on the normal to the plane of the coverslips. When the supporting lipid is in the L(beta') phase the helices are rigid on the timescale of (13)C-NMR. The configuration of gramicidin A is unaltered by L(alpha)-L(beta') phase transition of the bilayer lipid.