Short messenger RNAs (mRNAs) with hexitol residues in two codons were constructed and their properties were studied in an Escherichia coli in vitro translation system. The replacement of the natural ribonucleotides of mRNA in the AUG start codon and the UUC second codon by hexitol nucleotides did not influence the main steps of translation, as indicated by the same level of binding of mRNA with or without hexitol residues under P-site conditions, and the same yield of tRNA binding to the P- and A-sites. Moreover, both peptide formation and translocation took place on mRNAs with hexitol residues. The presence of an A-type messenger hexitol nucleic acid (mHNA)-transfer RNA (tRNA) duplex is important for efficient translation and the 2'-OH function in mRNA is not necessary for binding and movement through the ribosome. Groove shape recognition of the codon-anticodon complex, more than hydrogen-bond interactions of ribose residues in mRNA, is an important factor for correct translation.