A unique ribonuclease named Biota orientalis ribonuclease (Biota orientalis RNase) is purified to homogeneity from mature seeds of oriental arborvitae (Biota orientalis). The molecular mass of Biota orientalis RNase is about 13 kDa. When the concentration of Mg(2+) is 25 mM in the incubation buffer, the ribonuclease specifically cleaves the phosphodiester bond between C4453 and A4454 in region K (a region in domain VII) of 28S RNA in rat ribosome, resulting in inactivation of ribosome. Thus, it is a ribotoxin similar to alpha-sarcin. The region around C4453-A4454 in rat 28S rRNA is named "Biota orientalis RNase region." Rat ribosome treated by Biota orientalis RNase produces a small RNA fragment (S-fragment) that contains 333 nucleotides from the 3'-terminus of rat 28S rRNA. The distance between the cleavage-sites of alpha-sarcin (G4325) and Biota orientalis RNase (C4453) is 128 nucleotides. Under restricted conditions (25 mM Mg(2+)), the substrate specificity of Biota orientalis RNase is extremely high: it acts only on the "Biota orientalis RNase region" of the largest RNA in ribosomes from certain eukaryotes. The ribosome specifically damaged by Biota orientalis RNase is unable to EF-1alpha-dependently bind aminoacyl-tRNA, whereas the formation of the EF-2/GDP/ribosome complex is not affected. It is proposed that Biota orientalis RNase inactivates ribosome at least partially by interfering with the EF-1alpha-dependent binding of aminoacyl-tRNA to ribosome. Biota orientalis RNase might be a useful tool in studying the structure/function of ribosome.