Significance Despite the well-characterized structural symmetry of the dimeric transmembrane antibiotic gramicidin A, we show that the symmetry is broken by selective hydrogen bonding between eight waters comprising a transmembrane water wire and a specific subset of the 26 pore-lining carbonyl oxygens of the gramicidin A channel. The ¹⁷ O NMR spectroscopic resolution of the carbonyl resonances from the two subunits required the use of a world record high field magnet (35.2 T; 1,500 MHz for ¹ H). Uniquely, this result documented the millisecond timescale stability of the water wire orientation within the gramicidin A pore that had been reported to have only subnanosecond stability. These ¹⁷ O spectroscopic results portend wide applications in molecular biophysics and beyond.