Tail-anchored (TA) proteins are a class of polypeptides integrated into the membrane by a C-terminally located hydrophobic sequence which are present in all three domains of life. Proteins of this class lack an N-terminal signal peptide and reach their destination within the cell by posttranslational mechanisms. TA proteins perform a variety of essential functions on the cytosolic face of cellular membranes and, in several cases, determine the organelle identity. Some TA proteins insert directly into the lipid bilayer without the help of molecular machinery, suggesting that they may be ancestral proteins able to recruit lipids, contributing to the formation of intracellular compartments during cell evolution. Relevant progress has been made in recent years on the identification of TA protein sorting and the posttranslational translocation machineries. Interestingly, membrane lipid components were also found to be involved in the insertion mechanism. A bioinformatic approach is used to produce a catalogue of putative TA proteins encoded by the Arabidopsis thaliana genome, and intracellular localization is predicted based on features of well-characterized TA proteins. A recent strategy aimed at improving the accumulation of recombinant proteins expressed in transgenic plants is also discussed.