The effects of pH and temperature on the 300‐MHz 1H‐nmr spectra of three cardioactive polypeptides from sea anemones, anthopleurin‐A from Anthopleura xanthogrammica (AP‐A) and Anemonia sulcata toxins I and II (ATX I and II), are described. AP‐A and ATX II exhibit major spectral heterogeneity. Evidence from the pH and temperature studies and from high performance liquid chromatography indicates that this heterogeneity is conformational rather than chemical in origin. By contrast, purified isotoxins of ATX I show no evidence of conformational heterogeneity. The pKa values of most of the ionizable groups in these polypeptides are not strongly perturbed by interactions in the tertiary structure, with the exception of one of the Asp carboxylates, which has a pKa of ≲ 2 in AP‐A and ATX II and 3.0 in ATX I. Protonation of this carboxylate, suggested to be Asp‐9, leads to a conformational change in all three molecules. All three polypeptides are thermally stable, showing some conformational changes but not major unfolding at elevated temperatures.