The relationship between the process of rat brain protein synthesis inhibition by restrictocin and mitogillin and the induction of a specific cleavage in 28S rRNA has been examined. Restrictocin or mitogillin at a concentration of 6 nM inhibits protein synthesis to a level of 80%. The inhibition induced by 6 nM alpha-sarcin is of the same order, indicating that those molecules all catalytically inhibit protein synthesis. When restrictocin or mitogillin was reduced and alkylated, a 100-fold higher concentration of these chemically modified molecules was needed in order to obtain the same inhibition as with native molecules, suggesting a close correlation between activity and configuration of the inhibitors. The inhibitory activity of restrictocin or mitogillin was completely lost after oxidation with performic acid. The inhibition of protein synthesis was always correlated with the production of a fragment from the 3'-end of the 28S rRNA. This rRNA fragment had the same electrophoretic mobility as that produced by alpha-sarcin. The 5'-end sequence of the rRNA fragment produced by restrictocin, mitogillin or alpha-sarcin is AGGAA, demonstrating that restrictocin and mitogillin inhibit protein synthesis in the same way as does alpha-sarcin. The conservation of this region of the rRNA from archaebacteria to eukaryotic ribosomes indicates its importance in the elongation process.