The effect of interferon has been studied in a mixed cell-free protein synthesizing system. Hemoglobin (Hb) and Encephalomyocarditis virus (EMC)-RNA can be efficiently translated in vitro in a system containing S-30 lysates or run-off ribosomes from primary chick embryo fibroblasts (CEF) and a postmicrosomal supernatant from mouse ascites cells or a ribosomal-wash preparation from rabbit reticulocytes. Ribosomes prepared from CEF pretreated with high doses of homologous interferon (500 units/ml) were able to translate Hb-RNA in the presence of heterologous factors with the same efficiency as ribosomes prepared from control cells. Translation of EMC-RNAP WAS SLIGHTLY Reduced if ribosomes from interferon-treated cells were used in the mixed cell-free system, confirming previous reports. No inhibitory effect caused by interferon treatment of CEF cells could be detected on in vitro translation of natural mRNAs if the cells had, in addition to interferon treatment, been infected with vaccinia virus. Possible reasons for the different observations made with out cell-free protein synthesizing system from CEF and with cell-free systems prepared from mouse cells are discussed.