When the central valine residues 6, 7, and 8 of gramicidin A (gA) are shifted by one position, the resulting [Val(5), D-Ala(8)]gA forms right-handed channels with a single-channel conductance and average duration somewhat less than gA channels. The reduction in channel duration has been attributed to steric conflict between the side chains of Val(1) and Val(5) in opposing monomers (Koeppe, R. E. II, D. V. Greathouse, A. Jude, G. Saberwal, L. L. Providence, and O. S. Andersen. 1994. J. Biol. Chem. 269:12567-12576). To investigate the orientations and motions of valines in [Val(5), D-Ala(8)]gA, we have incorporated (2)H labels at Val 1, 5, or 7 and recorded (2)H-NMR spectra of oriented and nonoriented samples in hydrated dimyristoylphosphatidylcholine. Spectra of nonoriented samples at 4 degrees C reveal powder patterns that indicate rapid side chain "hopping" for Val(5), and an intermediate rate of hopping for Val(1) and Val(7) that is somewhat slower than in gA. Oriented samples of deuterated Val(1) and Val(7) show large changes in the methyl and C(beta)-(2)H quadrupolar splittings (Deltanu(q)) when Ala(5) in native gA is changed to Val(5). Three or more peaks for the Val(1) methyls with Deltanu(q) values that vary with the echo delay, together with an intermediate spectrum for nonoriented samples at 4 degrees C, suggest unusual side chain dynamics for Val(1) in [Val(5), D-Ala(8)]gA. These results are consistent with a steric conflict that has been introduced between the two opposing monomers. In contrast, the acylation of gA has little influence on the side chain dynamics of Val(1), regardless of the identity of residue 5.