Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this cotranslational mechanism. Recent genome sequencing and annotation studies highlighted over 2x104 putative peptide deformylase sequences. Furthermore, unpredicted modified bacterial PDF genes have been retrieved from many phages. Sequence comparisons with other known PDFs reveal that viral PDFs are devoid of the key ribosome-interacting C-terminal region. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity in vitro or in vivo. This investigation determines the activity of the recombinant Vp16 PDF in E. coli PDF defective cell to remove the N-formyl group at the protein N-terminus.