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Benthic dinoflagellates of the genusOstreopsisSchmidt are common in tropical and subtropical water, and some species produce toxins potentially involved in human intoxication events. A benthic bloom ofOstreopsis mascarenensisQuod was observed near Rodrigues Island during a survey of benthic dinoflagellates in the southwestern Indian Ocean. The morphology ofO. mascarenensiswas studied by LM and SEM. Preliminary screening of a crude extract of anO.mascarenensisbloom revealed neurotoxicity in mice similar to that induced by palytoxin. After partition of the crude extract, the highest toxicity was retained in the butanol-soluble fraction, which retained hemolytic activity suggestive of palytoxin analogues. Two new toxins, mascarenotoxin-A and -B, were resolved from this fraction by HPLC coupled to a diode array detector. The closed mass spectrum profile and fragmentation pattern obtained by advanced nano–electrospray ionization quadrupole time-of-flight mass spectrometry between purified toxins and a reference palytoxin confirmed the mascarenotoxins as palytoxin analogues. These results were confirmed by tandem mass spectrometry with the identification of specific fragment ionm/z327. An on-line liquid chromatography protocol coupled to tandem mass spectrometry was developed for detection of these palytoxin analogues. The present study describes the first purification, chemical, and toxicological characterization of new palytoxin analogues isolated from a benthic bloom ofO. mascarenensis. These results suggest thatO. mascarenensis, which is largely distributed in the southwestern Indian Ocean, could be a source of palytoxin poisoning in this tropical area. [ABSTRACT FROM AUTHOR]