C-tail-anchored (TA) proteins constitute a heterogeneous group of membrane proteins that are inserted into membranes by unique post-translational mechanisms and that play key roles within cells. During recent years, bioinformatic screens on eukaryotic genomes have helped to obtain comprehensive pictures of the number, intracellular distribution and functions of TA proteins, but similar screens had not yet been carried out on prokaryotic cells. Here, we report the results of a bioinformatic screen of the genomes of two bacteria and one archeon. We find that all three of these prokaryotes contain TA proteins in proportions approaching those found in eukaryotic cells, indicating that this protein group is present in all three domains of life. Although some of our hits correspond to proteins of unknown function, others are enzymes with hydrophobic substrates or have functions carried out at the inner face of the cytoplasmic membrane. To generate hypotheses on the insertion mechanisms of prokaryotic TA proteins, we compared the sequences of the prokaryotic and eukaryotic versions of Asna1/Trc40/GET3, a cytosolic ATPase that plays a key role in TA protein post-translational delivery to membranes in eukaryotic cells. We found that hydrophobic residues involved in TA binding by the eukaryotic chaperone (Mateja et al., Nature 2009;461:361–366) are generally replaced with equally hydrophobic amino acids in the archeal homologue (ArsA), whereas this is not the case for the bacterial protein. Thus, eukaryotes may have inherited the GET3 targeting pathway from our archeal ancestor, while the bacterial homologue may be exclusively dedicated to heavy metal resistance. [ABSTRACT FROM AUTHOR]