ShK toxin from the sea anemone Stichodactyla helianthus is a 35-residue protein that binds to the Kv1.3 ion channel with high affinity. Recently we determined the X-ray structure of ShK toxin by racemic crystallography, in the course of which we discovered that D-ShK has a near-background IC50 value ~50,000 times lower than that of the L-ShK toxin. This lack of activity was at odds with previously reported results for an ShK diastereomer designated D-allo-ShK, for which significant biological activity had been observed in a similar receptor-blocking assay. As reported, D-allo-ShK was made up of D-amino acids, but with retention of the natural stereochemistry of the chiral side chains of the Ile and Thr residues, i.e. containing D-allo-Ile and D-allo-Thr along with D-amino acids and glycine. To understand its apparent biological activity, we set out to chemically synthesize D-allo-ShK and determine its X-ray structure by racemic crystallography. Using validated allo-Thr and allo-Ile, both L-allo-ShK and D-allo-ShK polypeptide chains were prepared by total chemical synthesis. Neither the L-allo-ShK nor the D-allo-ShK polypeptides folded, whereas both L-ShK and D-ShK folded smoothly under the same conditions. Re-examination of NMR spectra of the previously reported D-allo-ShK protein revealed that diagnostic Thr and Ile signals were the same as for authentic D-ShK. On the basis of these results, we conclude that the previously reported D-allo-ShK was in fact D-ShK, the true enantiomer of natural L-ShK toxin, and that the apparent biological activity may have arisen from inadvertent contamination with trace amounts of L-ShK toxin.