The complete sequence-specific assignments of resonances in the 1H-NMR spectrum of huwentoxin-I from the Chinese bird spider, Selencocosmia huwena, is described. A combination of two-dimensional NMR experiments including 2D-COSY, 2D-NOESY, and 2D-TOCSY has been employed on samples of the toxin dissolved in D2O and in H2O for assignment purposes. Protons belonging to spin systems for each of the 33 amino acids were identified. The sequence-specific assignments were facilitated by the identification of d alpha N connectivities on the fingerprint regions of the COSY and NOESY spectra and were supported by the identification of dNN and d alpha N connectivities in the TOCSY and NOESY spectra. These studies provide a basis for the determination of the solution-phase conformation of this toxin.