AIM To study the bufadienolides in the Chinese traditional drug "Ch'an Su" and their cytotoxic activity. METHOD Various chromatographic techniques were used to isolate the constituents, and their structures were elucidated through physical and spectroscopic data. RESULTS Twenty compounds were isolated, and eighteen were evaluated in vitro for their cytotoxic activity against A-549 and K-562 cells. CONCLUSION Compound 1 (bufalin 3β-acrylic ester) was a new bufadienolide and exhibited the most potent activity against the two tumor cell lines with IC50 values of 7.16 and 6.83 nmol · L(-1). The relationships between structure and activity are discussed.