The purpose of this study was to characterize DON-induced rRNA fragmentation with respect to (1) specific sites of 18S and 28S rRNA cleavage and (2) identity of upstream signaling events that mediate this process. The results demonstrate that DON promotes cleavage of 18S rRNA and 28S rRNA into a minimum of five and six fragments, respectively. Furthermore, DON-induced rRNA cleavage occurred concurrently with apoptosis was mediated by sequential activation of PKR, Hck, p38, p53, and several caspases. Three other translational inhibitors, satratoxin G (SG), anisomycin, and ricin, evoked comparable selective rRNA cleavage to DON, suggesting that a common mechanism might exist for other ribotoxins.