Site-directed mutagenesis of trichosanthin (TCS), a ribosome inactivating protein with a broad spectrum of biological activities, was carried out to ascertain the importance of the Glu 160 and Glu 189 residues to the protein synthesis-inhibitory, antiproliferative, immunosuppressive and embryotoxic activities of TCS. Replacement of Glu 160 with alanine and with aspartate produced muteins, designated [E160A] and [E160D] respectively, with considerably attenuated protein synthesis-inhibitory, antiproliferative, immunosuppressive and embryotoxic activities, indicating that Glu 160 in TCS plays a role in its biological activity. [E160A] was, however, more potent than [E160D] because in the former mutein, Glu 189 constitutes a back-up of the carboxylate group but in the latter mutein, the negative charge from Asp is at a suboptimal position. The mutein [E160A, E189A] formed by mutation of both Glu 160 and Glu 189 retained considerable embryotoxic activity, suggesting that other amino acids in the active site were able to partially replace the role of Glu 160 and Glu 189. The TCS muteins also exhibit higher toxicity toward cultured embryos than cultured cells (spleen cells and tumor cells).