Membrane proteins fulfill a number of vital functions in prokaryotic and eukaryotic cells. They are often organized in multicomponent complexes, folded within the membrane bilayer and interacting with the cytoplasmic and periplasmic or external soluble compartments. For the biogenesis of integral membrane proteins, the essential biochemical steps are (1) the insertion and topogenesis of the transmembrane protein segments into the lipid bilayer, (2) the three-dimensional folding of the translocated hydrophilic domains, and (3) the assembly into multimeric complexes. Intensive research has elucidated the basic mechanisms of membrane protein insertion in the homologous translocation machineries of different cellular systems. Whereas the Sec translocation system is found in the endoplasmic reticulum of eukaryotic cells and in the prokaryotic plasma membrane, the YidC-Oxa1 membrane insertase is present in prokaryotic and organellar membranes. This review focuses on the discoveries of the YidC system in bacterial as well as the Oxa1/Alb3 protein family of eukaryotic cells and will particularly emphasize evolutionary aspects.