D-galactosamine produces an early defect in protein synthesis, independent of its effects on RNA synthesis. Here we show that the defect in protein synthesis is inherent in purified ribosomal subunits in vitro. Further, galactosamine treatment is associated with an 85% decrease in methylation of ribosomal RNA, involving all sites (2'-0-ribose and base positions), intact ribosomes from galactosamine-treated animals can be methylated to a greater extent than control ribosomes and in vitro methylation restores their functional capacity. Statistical analyses of these data, along with those with a number of other hepatotoxins, reveal a correlation coefficient of r = 0.95 (p less than 0.003) between protein synthetic capacity and ribosomal RNA methylation, and linear regression accounts for more than 90% of the observed variation. In contrast, no relationship was found between nucleolar RNA methylation and protein synthetic capacity. A relationship of borderline statistical significance was found between messenger RNA methylation and protein synthetic capacity, but it does not appear consistent with results obtained after CCl4 intoxication. These results lend strong support to the notion that methylation status of ribosomal RNA is an important control for protein synthesis in quiescent hepatocytes and that net hypomethylation is a common response to such divergent hepatotoxins as CCl4, ethionine and D-galactosamine.