Abstract Under conditions which are optimal for RNA-directed polypeptide chain initiation in vitro, ribosomes bind to single-stranded DNA and synthesize fMet-dipeptides. The products directed by natural single-stranded or denatured double-stranded DNA from eight different organisms are strikingly similar: predominantly fMet-Thr, fMet-Val and fMet- Leu Ile. For four of these templates, there exists evidence that these dipeptides are not representative of those synthesized from the corresponding messenger RNAs. The observations are discussed in the light of results which indicate that DNA can serve accurately as a substitute message for binding of ribosomes and synthesis of polypeptides in. vitro.