Homonuclear two-dimensional and three-dimensional 1H-NMR spectroscopy has been utilized to study the 15.9-kDa protein soybean leghemoglobin. NMR experiments were performed on the diamagnetic carbon monoxide complex at two temperatures and two pH values. Sequence-specific assignments have been made for 94% of the backbone and approximately 70% of the expected side-chain resonances. The secondary structure of leghemoglobin in solution has been determined on the basis of NOE connectivity patterns, hydrogen exchange and chemical-shift analyses. Leghemoglobin consists of seven helices and, unlike mammalian myoglobins, is missing the D helix. Instead an extended loop, the CE loop, is observed which might have importance for ligand entry into and exit from the protein interior. The hydrogen exchange behavior for the F helix and at the beginning of the A helix suggests different dynamic stability compared to other helical regions in leghemoglobin. Population of a second protein conformation, in which there is perturbation at the A-G-H helix interface, is observed at low pH.