Polyadenylation is a posttranscriptional modification present throughout all the kingdoms of life with important roles in regulation of RNA stability, translation, and quality control. Functions of polyadenylation in prokaryotic and organellar RNA metabolism are still not fully characterized, and poly(A) tails appear to play contrasting roles in different systems. Here we present a general overview of the polyadenylation process and the factors involved in its regulation, with an emphasis on the diverse functions of 3' end modification in the control of gene expression in different biological systems.