The effect of the duration of interferon treatment of mouse L929 fibroblasts on the expression of ribosome-associated and cell-sap-localized translation inhibition, protein phosphorylation, and messenger RNA (mRNA) methylation and degradation was investigated. The following results were obtained: (1) Ribosomal salt-wash fractions prepared from L929 cells treated for 12 or 18 hr significantly inhibited the translation of methylated reovirus mRNA catalyzed by the cell-free system prepared from untreated ascites tumor cells. The translation of reovirus mRNA was slightly stimulated by the ribosomal salt-wash fractions prepared from untreated cells and cells treated for 2 hr and was slightly inhibited by the salt-wash fraction from cells treated for 6 hr. (2) Cell-sap fractions prepared from untreated cells and from cells treated for 2 or 6 hr did not appreciably affect the translation of reovirus mRNA in the absence of double-stranded RNA (dsRNA); cell-sap fractions prepared from cells treated for 12 or 18 hr were slightly inhibitory. (3) Neither reovirus genome dsRNA nor polyriboinosinic acid-polyribocytidylic acid inhibited the translation of reovirus mRNA by the untreated ascites system. The inhibitory activity of ribosomal salt-wash fractions from interferon-treated cells was dsRNA independent, whereas dsRNA enhanced the inhibitory activity of cell-sap fractions from interferon-treated cells. (4) Interferon treatment significantly enhanced a cell-sap-independent phosphorylation of at least three proteins present in the ribosomal salt-wash fractions. Phosphorylation of an approximately 50,000-dalton component was maximally enhanced after 12 hr of interferon treatment and was increased by dsRNA; phosphorylation of 30,000- and <20,000-dalton components was enhanced earlier and was dsRNA independent. (5) An increase in the nuclease-mediated degradation of [3H]uridine-labeled methylated reovirus mRNA catalyzed by ribosomal salt-wash fractions was detectable after 6 hr of interferon treatment and was maximally enhanced after 12 hr of interferon treatment to about threefold the level of the untreated ribosomal salt-wash fraction. The degradation was not enhanced by dsRNA. The levels of reovirus mRNA degradation catalyzed by cell-sap fractions from untreated cells and from cells treated with interferon for 2 to 18 hr were comparable. (6) No significant difference was observed in the ability of cell-sap fractions from untreated cells or from cells treated with interferon for 2 to 18 hr to catalyze the methylation of unmethylated reovirus mRNA. In addition, none of the ribosomal salt-wash fractions significantly inhibited reovirus mRNA methylation catalyzed by untreated ascites cell-free extracts. These results suggest that interferon treatment may mediate multiple biochemical changes in murine cells, including the specific phosphorylation of protein(s) associated with ribosomes that possess interferon-mediated inhibitor(s) of viral mRNA translation.