We demonstrate a general nuclear magnetic resonance (NMR) spectroscopic approach in obtaining high-resolution O-17 (spin-5/2) NMR spectra for biological macromolecules in aqueous solution. This approach, termed quadrupole central transition (QCT) NMR, is based on the multiexponential relaxation properties of half-integer quadrupolar nuclei in molecules undergoing slow isotropic tumbling motion. Under such a circumstance, Redfield's relaxation theory predicts that the central transition, m(1) = +1/2 <-> -1/2, can exhibit relatively long transverse relaxation time constants, thus giving rise to relatively narrow spectral lines. Using three robust protein-ligand complexes of size ranging from 65 to 240 kDa, we have obtained O-17 QCT NMR spectra with unprecedented resolution, allowing the chemical environment around the targeted oxygen atoms to be directly probed for the first time. The new QCT approach increases the size limit of molecular systems previously attainable by solution O-17 NMR by nearly 3 orders of magnitude (1000-fold). We have also shown that, when both quadrupole and shielding anisotropy interactions are operative, O-17 QCT NMR spectra display an analogous transverse relaxation optimized spectroscopy type behavior in that the condition for optimal resolution depends on the applied magnetic field. We conclude that, with the currently available moderate and ultrahigh magnetic fields (14 T and higher), this O-17 QCT NMR approach is applicable to a wide variety of biological macromolecules. The new O-17 NMR parameters so obtained for biological molecules are complementary to those obtained from H-1, C-13, and N-15 NMR studies.