Buthotus judaicus toxin 1 (BjTx-1) and toxin 2 (Bjtx-2), two novel peptide activators of ryanodine receptors (RyR), were purified from the venom of the scorpion B. judaicus. Their amino acid sequences differ only in one residue out of 28 (residue 16 corresponds to Lys in BjTx-1 and Ile in BjTx-2). Despite a slight difference in EC₅₀, both toxins increased binding of [³H]ryanodine to skeletal SR at micromolar concentrations, but had no effect on cardiac or liver microsomes. Their activating effect was Ca²⁺-dependent and was synergized by caffeine. Bj toxins also increased binding of [³H]ryanodine to the purified RyR1, suggesting that a direct protein-protein interaction mediates the effect of the peptides. BjTx-1 and BjTx-2 induced Ca2+ release from Ca²⁺-loaded SR vesicles in a dose-dependent manner and induced the appearance of long-lived subconductance states in skeletal RyRs reconstituted into lipid bilayers. Three-dimensional structural modeling reveals that a cluster of positively charged residues (Lys11-Lys16) is a prominent structural motif of both toxins. A similar structural motif is believed to be important for activation of RyRs by Imperatoxin A (IpTx_a), another RyR-activating peptide (Gurrola et al, 1999, J. Biol. Chem. 274, 7879). Thus, it is likely that Bj toxins and IpTx_a bind to RyRs by means of electrostatic interactions that lead to massive conformational changes in the channel protein. The different affinity and structural diversity of this family of scorpion peptides makes them excellent peptide probes to identify RyR domains that trigger the channel to open.