Eighteen synthetic analogs of ShK toxin, a thirty-five residue K channel blocker derived from the sea anemone Stichodactyla helianthus, were prepared in order to identify functionally important residues. CD spectra of sixteen of the analogs were virtually identical with the spectrum of wild-type toxin, indicating that the conformations were not affected by the substitutions. A conserved residue, Lys22, is essential for ShK binding to rat brain K channels which are primarily of the Kv1.2 type. However, a cationic side chain at position 22 is not essential for binding to the human Jurkat T-lymphocyte Kv1.3 channel. While decreasing bulkiness at this position affected toxin affinity for the brain K channels, increasing bulkiness decreased toxin affinity for both brain and lymphocyte K channels. In contrast to the rat brain channels, ShK binding to Kv1.3 was sensitive to substitution at Lys9 and Arg11.