AbstractGPI lipid anchoring is an important post‐translational modification of eukaryote proteins in the endoplasmic reticulum. In total, 19 genes have been directly implicated in the anchor synthesis and the substrate protein modification pathway. Here, the molecular functions of the respective proteins and their evolution are analyzed in the context of reported literature data and sequence analysis studies for the complete pathway (http://mendel.imp.univie.ac.at/SEQUENCES/gpi‐biosynthesis/) and questions for future experimental investigation are discussed. Studies of two of these proteins have provided new mechanistic insights. The cytosolic part of PIG‐A/GPI3 has a two‐domain α/β/α‐layered structure; it is suggested that its C‐terminal subsegment binds UDP‐GlcNAc whereas the N‐terminal domain interacts with the phosphatidylinositol moiety. The lumenal part of PIG‐T/GPI16 apparently consists of a β‐propeller with a central hole that regulates the access of substrate protein C termini to the active site of the cysteine protease PIG‐K/GPI8 (gating mechanism) as well as of a polypeptide hook that embraces PIG‐K/GPI8. This structural proposal would explain the paradoxical properties of the GPI lipid anchor signal motif and of PIG‐K/GPI8 orthologs without membrane insertion regions in some species. BioEssays 25:367–385, 2003. © 2003 Wiley Periodicals, Inc.