A simple method for depleting E. coliS30 extracts of endogenous tRNA has been developed. An $ethanolamine-Sepharose^{(R)}$ column equilibrated with water selectively captured the tRNA molecules in E. coli S30 extracts. As a result, S30 extracts filtered through this column became completely dependent upon the addition of exogenous tRNA to mediate cell-free protein synthesis reactions. We anticipate that the procedures developed and described will be particularly useful for in vitro suppression reaction studies designed to introduce unnatural amino acids into protein molecules.