To understand the events that occur in the early stages of the folding of hUBF HMG box 1, we characterized its pH 2.1 unfolded state in detail with NMR. Through a triple resonance strategy, the assignments of complete backbone and some side chains were achieved. Then, significant conformational information was extracted from secondary chemical shifts, interresidual (1)H-(1)H NOEs, (3)J(HNHA) coupling constants, amide proton temperature coefficients, and (15)N relaxation data. The secondary chemical shifts for (13)CA, (13)CB, (13)CO, (1)HA, and (1)HN indicate that the residues between 64 and 78 exhibit a substantial preference for helical structure in the acid-unfolded state, which is also evidenced by the relatively more negative deviations of (3)J(HNHA) and amide proton temperature coefficients from their corresponding random-coil values and particularly confirmed by the strongest sequential d(NN)(i, i + 1) proton NOEs along the region. Following this region until residue 82 is a segment that tends to form a turn-like structure, which is unstable and exchanges between alternative states. In addition, some evidences imply that the regions 18-28 and 38-43 also possess propensities for helical structure but to a different less degree than the region 64-78. The polypeptide backbone dynamics investigated using reduced spectral density function shows apparent motional restrictions in residual structural regions and to less extent at some hydrophobic residues. On the basis of the results presented herein, we propose a potential protein-folding pathway on which these residual structures play a role of initiation site in the early folding stages.