Evaluation of positive and negative ion fast atom bombardment (FAB) mass spectrometry for structural investigations have been studied on 30 cardiac glycosides in comparison with their field desorption (FD) mass spectra. FD mass spectra showed the prominent molecular cluster ions and the same protonation induced fragments as that established for steroid oligoglycosides. Even if the molecular cluster ions of some di- and triglycosides were hardly detected in the positive ion FAB mass spectra owing to their low intensities, a dramatic improvement was observed when sodium chloride or potassium iodide was added to the glycerol matrix. Furthermore, the prominent molecular ion species, [M-H]-, and the sequence of glycosidic linkage were easily detected in the negative mode. The combination of positive and negative ion FAB mass spectrometry of the cardenolide-type cardiac glycosides provides useful information concerning the molecular weight as well as the structures of both aglycone and sugar moieties.