Elements of secondary structure in the sea anemone polypeptides anthopleurin A and Anemonia sulcata toxin I have been defined with the following nuclear magnetic resonance (NMR) spectroscopic data: the pattern of nuclear Overhauser enhancement (NOE) connectivities observed in two-dimensional NMR spectra for protons along the polypeptide backbone, NOE's between protons on separate strands of the polypeptide backbone, peptide NH exchange rates, and NH-Hα spin-spin coupling constants. These two polypeptides contain a region of four short strands of antiparallel β-sheet but little or no α-helix. This region of β-sheet brings the aromatic rings of Trp-23 and -33 into close proximity to form the nucleus for a small hydrophobic region. A type II reverse turn involving residues 30–33 has also been defined. Our results are compared with previous predictions of the secondary structure of these polypeptides. The structures are also discussed in relation to that of a scorpion toxin that appears to bind to a similar site on the sodium channel of excitable tissue.