We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3'-rapid amplification of cDNA ends and 5'-rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date. Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC(50) value of 0.38 microm in rat cardiac myocytes. Different from scorpion beta-toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10 microm eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion alpha-like toxin) completely. JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.