Naturally occurring toxins are invaluable tools for exploration of the structure and function relationships of voltage-gated sodium channels (VGSCs). In this study, we isolated and characterized a novel VGSC toxin named jingzhaotoxin-II (JZTX-II) from the tarantula Chilobrachys jingzhao venom. JZTX-II consists of 32 amino acid residues including two acidic and two basic residues. Cloned and sequenced using 3'- and 5'-rapid amplification of the cDNA ends, the full-length cDNA for JZTX-II was found to encode a 63-residue precursor which contained a signal peptide of 21 residues, a propeptide of 10 residues and a mature peptide of 32 residues. Under whole-cell voltage-clamp conditions, JZTX-II significantly slowed rapid inactivation of TTX-resistant (TTX-R) VGSC on cardiac myocytes with the IC50=0.26+/-0.09 microM. In addition, JZTX-II had no effect on TTX-R VGSCs on rat dorsal root ganglion neurons but exerted a concentration-dependent reduction in tetrodotoxin-sensitive (TTX-S) VGSCs accompanied by a slowing of sodium current inactivation similar to delta-ACTXs. It is notable that TTX-S VGSCs on cultured rat hippocampal neurons were resistant to JZTX-II at high dose. Based on its high selectivity for mammalian VGSC subtypes, JZTX-II might be an important ligand for discrimination of VGSC subtypes and for exploration of the distribution and modulation mechanisms of VGSCs.