Further examination of the tunicate extract resulted in isolation of a total of 26 ritterazines whose structures were elucidated by spectroscopic analyses. Their cytotoxicity against P388 leukemia cells and that of derivatives obtained from the most active ritterazine B led to some structure-activity relationships. Ritterazine B which was unsymmetric was the most potent cytotoxin; ritterazines A,D,E,F,G,H,I,J,K,L,M and Y were also highly cytotoxic. These compounds have the 5/6 spiro ring and 12',25'-diol functionalities in common in the western hemisphere. However ritterazines N,O,P,Q,R,S,W,X, and Z having no 5/6 spiroketal were marginally active. Therefore the 5/6 spiroketal was supposed to be a important function for the expression of cytotoxicity, because the cleavage of the 5/6 spiroketal decreased their activity. Furthermore ritterazine T which had no conventional steroidal skeleton with OH-12 in the eastern hemisphere, but the 5/6 spiro ring in the western hemisphere showed weak cytotoxicity. Therefore, the presence of a 5/6 spiro ring in one end, a 5/5 spiro ring in the other end, and the hydroxyl group on C12 are shown to be important for cytotoxic activity. However acetylation of secondary hydroxyl groups gave interesting information. Compared with oxidation, acetylation of OH-12 does not decrease cytotoxicity, but induction of acetyl groups in the western hemisphere showed weaker activity. It is possible to consider that the steric hindrance by acetyl groups in the western hemisphere influences the expression of the cytotoxicity. In the future these findings will stimulate research on the mode of action of this important class of compounds.