We have developed a simple and effective method for isolating pectenotoxin-2 (PTX-2) from Dinophysis cells collected from a natural bloom. A two-step extraction procedure followed by two column chromatography steps produced PTX-2 in high purity suitable for use as an analytical standard and for toxicological studies. Incubation of purified PTX-2 with the supernatant from ultracentrifuged blue ( Mytilus edulis) or Greenshell ® ( Perna canaliculus) mussel hepatopancreas homogenate caused rapid conversion to pectenotoxin-2 seco acid (PTX-2 SA). Purification of PTX-2 SA was achieved by solvent extraction followed by column chromatography. PTX-2 and PTX-2 SA were fully characterized by LC–MS and NMR, and full 1H and 13C NMR assignments were obtained. Okadaic acid C 8-diol ester was isolated during the purification of PTX-2, and its identity confirmed by NMR and LC–MS analyses. Pectenotoxin-2 seco acid methyl ester, identified by LC–MS, was also produced during the hydrolytic procedure due to the presence of methanol. PTX-2 was acutely toxic to mice by i.p. injection (LD 50=219 μg/kg) but no effects were seen with PTX-2 SA at 5000 μg/kg. Neither PTX-2 nor PTX-2 SA was overtly toxic to mice by the oral route at doses up to 5000 μg/kg. No diarrhea was observed in mice dosed with either compound, suggesting that pectenotoxins do not belong in the diarrhetic shellfish poison group.