The effect of interferon (IFN) on the adsorption, penetration and subsequent binding of the incoming genome RNA of Moloney murine leukemia virus (MLV) to polyribosomes, was studied in NIH/3T3 mouse fibroblasts. Virus adsorption was assayed by determining reverse transcriptase activity in the inoculating virus stock and in the cell membrane fraction before and after 45 minutes of infection. Both measurements suggested that IFN had no effect on virus adsorption. Virus penetration was determined by measuring the amount of viral RNA in the cell cytoplasm at 45 minutes after infection. This amount was remarkably lower in IFN-treated than in untreated cells. This reduction was not due to inhibition of a possible induction of endogenous viral genetic information by the penetrating virions, but was proved to be a direct effect of IFN on virus penetration, which was related to the IFN-induced antiviral state. The effect of IFN on binding of parental genome RNA to polyribosomes was then investigated by analysing Crt hybridization kinetics of polyribosomal viral RNA at different time intervals after infection. While in untreated control cells maximal binding occurred at 3 hours postinfection, this maximal binding was observed in IFN-treated cells at 5 hours postinfection. The distribution of viral RNA molecules between sub-cytoplasmic fractions at 3 hours after infection was, in IFN-treated cells, significantly different from that observed in the untreated cells.