The structure and dynamics of equilibrium intermediate in the unfolding pathway of the human acidic fibroblast growth factor (hFGF-1) are investigated using a variety of biophysical techniques including multidimensional NMR spectroscopy. Guanidinium hydrochloride (GdnHCl)-induced unfolding of hFGF-1 proceeds with the accumulation of a stable intermediate state. The transition from the intermediate state to the unfolded state(s) is cooperative without the accumulation of additional intermediate(s). The intermediate state induced maximally in 0.96 M GdnHCl is found to be obligatory in the folding/unfolding pathway of hFGF-1. Most of the native tertiary structure interactions are preserved in the intermediate state. H-1-N-15 chemical shift perturbation data suggest that the residues in the C-terminal segment including those located in the P-strands M X, and XI undergo the most discernible structural change(s) in the intermediate state in 0.96 M GdnHCl. hFGF-1 in the intermediate state (0.96 M GdnHCl) does not bind to its ligand, sucrose octasulfate. Limited proteolytic digestion experiments and hydrogen-deuterium exchange monitored by N-15 heteronuclear single quantum coherence (HSQC) spectra show that the conformational flexibility of the protein in the intermediate state is significantly higher than in the native conformation. N-15 spin relaxation experiments show that many residues located in beta-strands IX, X, and XI exhibit conformational motions in the micro- to millisecond time scale. Analysis of N-15 relaxation data in conjunction with the amide proton exchange kinetics suggests that residues in the beta-strands II, VIII, and XII possibly constitute the stability core of the protein in the near-native intermediate state.