AbstractAlthough the first poly(A) polymerase (PAP) was discovered in Escherichia coli in 1962, the study of polyadenylation in bacteria was largely ignored for the next 30 years. However, with the identification of the structural gene for E. coli PAP I in 1992, it became possible to analyze polyadenylation using both biochemical and genetic approaches. Subsequently, it has been shown that polyadenylation plays a multifunctional role in prokaryotic RNA metabolism. Although the bulk of our current understanding of prokaryotic polyadenylation comes from studies on E. coli, recent limited experiments with Cyanobacteria, organelles, and Archaea have widened our view on the diversity, complexity, and universality of the polyadenylation process. For example, the identification of polynucleotide phosphorylase (PNPase), a reversible phosphorolytic enzyme that is highly conserved in bacteria, as an additional PAP in E. coli caught everyone by surprise. In fact, PNPase has now been shown to be the source of post‐transcriptional RNA modifications in a wide range of cells of prokaryotic origin including those that lack a eubacterial PAP homolog. Accordingly, the past few years have witnessed increased interest in the mechanism and role of post‐transcriptional modifications in all species of prokaryotic origin. However, the fact that many of the poly(A) tails are very short and unstable as well as the presence of polynucleotide tails has posed significant technical challenges to the scientific community trying to unravel the mystery of polyadenylation in prokaryotes. This review discusses the current state of knowledge regarding polyadenylation and its functions in bacteria, organelles, and Archaea. WIREs RNA 2011 2 256–276 DOI: 10.1002/wrna.51This article is categorized under: RNA Processing > 3' End Processing RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability