Upon ethyl nitrosourea treatment of RNA of tobacco mosaic virus, up to four phosphodiester groups may be alkylated per molecule without chain breakage, as shown be sucrose gradient centrifugation. This indicates that ribophosphotriesters are quite stable. However, when this alkylation reaction is of longer duration and 6 to 10 triesters are formed, then an average of 1 to 2 breaks occurs and little or no intact RNA can be isolated. Methyl nitrosourea is less effective in forming triesters (about 25% of total alkyl groups compared to about 65% for ethyl nitrosourea), and a greater number of alkyl groups can, therefore, be introduced before breaks occur. Diethyl sulfate and dimethyl sulfate, which alkylate almost only the bases of nucleic acids, do not cause significant degradation of RNA of tobacco mosaic virus, even when as many as 70 alkyl groups are bound. All types of alkylation cause similar losses in viral infectivity at low levels of alkylation. Thus, an average of two chemical events leads to one lethal event, regardless of the nature of the alkylating reaction, which, for example, is with dimethyl sulfate about 65% on the N-7 guanine while with ethyl nitrosourea it is about 65% on phosphodiesters. It is thus concluded that all alkyl groups, whether on the base or on the phosphate, have the same potential to cause inactivation and that inactivation of RNA can result from phosphotriester formation per se.