Tityus kappa (Ts κ), a novel toxin from the venom of the scorpion Tityus serrulatus, is a 35‐residue polypeptide cross‐linked by three disulphide bridges and acts on small‐conductance calcium‐activated potassium channels (SK channels). Ts κ was chemically synthesized using the solid‐phase method and characterized. The synthetic product, sTs κ, was indistinguishable from the natural toxin when tested in vitro in competition assay with radiolabelled apamin for binding to rat brain synaptosomes (IC₅₀ = 3 nm). The sTs κ was further tested in vivo for lethal activity to mice following intracerebroventricular inoculation (LD₅₀ = 70 ng per mouse). The half‐cystine pairings were formerly established by enzyme‐based cleavage of sTs κ; they were between Cys₇–Cys₂₈, Cys₁₃–Cys₃₃ and Cys₁₇–Cys₃₅, which is a disulphide bridge pattern similar to that of other short scorpion toxins. According to previous studies on SK channel‐acting toxins, the putative influence of certain basic residues of Ts κ (i.e. Arg₆, Arg₉, Lys₁₈, Lys₁₉) in its pharmacological activity was investigated using synthetic point‐mutated analogues of the toxin with an Ala substitution at these positions. Data from binding assay, together with conformational analysis of the synthetic analogues by ¹H‐NMR, suggest that Arg₆, and to a lesser extent Arg₉, are important residues for an high‐affinity interaction of this toxin with SK channels; interestingly these residues are located outside the α‐helical structure, whereas the pharmacologically important basic residues from other SK channel‐specific toxins had been located inside the α‐helix.