Two new polypeptide toxins named Hm-1 and Hm-2 were isolated from the venom of the crab spider Heriaeus melloteei. These toxins consist of 37 and 40 amino acid residues, respectively, contain three intramolecular disulfide bonds, and presumably adopt the inhibitor cystine knot motif. Hm-1 is C-terminally amidated and shows a low degree of homology to spider toxins agelenin and micro-agatoxin-II, whereas Hm-2 has no relevantly related peptide sequences. Hm-1 and Hm-2 were found to act on mammalian voltage-gated Na(+) channels. Both toxins caused a strong decrease of Na(+) current peak amplitude, with IC(50) values of 336.4 and 154.8 nM, respectively, on Na(V)1.4. Hm-1 and Hm-2 did not shift the voltage-dependence of activation, nor did they change the kinetics of fast inactivation of the Na(+) currents. Interestingly, both toxins negatively shifted the steady-state inactivation process, which might have important functional consequences in vivo. However, this hyperpolarizing shift cannot by itself explain the observed inhibition of the Na(+) current, indicating that the two presented toxins could provide important structural information about the interaction of polypeptide inhibitors with voltage-gated Na(+) channels.