Translation of R17 ribonucleic acid (RNA) by Escherichia coli cell-free extracts was examined by studying lysine incorporation into soluble tryptic peptides from the in vitro coat protein. A sensitive thin-layer peptide mapping technique showed that several minor peptides containing lysine were present in tryptic digests both of protein coat synthesized in vitro and of natural coat protein. Radioactive double labeling of these minor peptides showed that they arose from irregularities of tryptic cleavage during protein digestion rather than from errors in translation. Five unique lysyl tryptic peptide sequences were shown to contain essentially equivalent amounts of radioactivity when the lysine content of the minor peptides was taken into account in a quantitative estimation of total lysine incorporation in vitro. This result demonstrates a high fidelity of in vitro translation of R17 RNA in the E. coli cell-free system.