A new cytotoxic steroidal alkaloid, plakinamine I, with an unprecedented 3 alpha-amino-19-acetoxy nucleus was isolated from a Corticium sp. sponge. The structure has been elucidated by spectroscopic analysis and secured by the synthesis of the model 19-acetoxy-3 alpha-aminocholestane. The evaluation of the cytotoxic activities of several synthetic derivatives, intermediates of the above synthesis, allowed us to explore the influence of the different functions on the cytotoxicity observed for natural plakinamine I. (c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)