The sea anemone polypeptide anthopleurin-A (AP-A) at nanomolar concentrations enhances myocardial contractility without affecting automaticity. It has a therapeutic index higher than that of the digitalis glycosides, and may serve as a molecular model for designing a new class of inotropic drugs acting on the myocardial Na channel at site 3. AP-A is a 49 residue peptide crosslinked by three disulfide bonds; its tertiary structure has been determined by NMR. Here we report the solid-phase synthesis of this polypeptide. Synthetic AP-A displayed CD and NMR spectra identical with those of the natural toxin; it possessed 94 +/- 15% of the inotropic activity of natural AP-A. Therefore, it is feasible to prepare various type 1 sea anemone toxin analogs by solid-phase chemical synthesis in order to identify side chains important for peptide folding and interaction with sodium channels.