The protein-synthesizing S 30 extract of Escherichia coil contains tRNA, which limits its applications in cell-free protein synthesis. Here, we show that at least Arg- and Seracceptor activities can be removed from a standard S 30 extract by treatment with an immobilized RNase A resin. This RNase-treated extract exhibits no protein synthesis activity, but regains it when supplied with crude E. coli tRNA and a small amount of human placental RNase inhibitor. The protein synthesis is dependent on the addition of tRNA in the presence of the RNase inhibitor. Chloramphenicol acetyltransferase was synthesized with this system and found to be active.