Several pathways are compartmentalized in the endoplasmic reticulum (ER). These intraluminal activities require the passage of substrates, cofactors, and products through the ER membrane. The arguments for a general permeability of the ER membrane are contradicted by strong biochemical, pharmacological, clinical, and genetic evidence, which indicates that the lipid bilayer has a barrier function and that specific transport activities are needed in the membrane. Consequently, the ER lumen can be regarded as a separate metabolic compartment. This article overviews the best characterized intraluminal processes in which the compartmentation is important either by defining an intraluminal milieu, by limiting the rate of the reaction, by determining the specificity, or by creating a common substrate pool because of the colocalization.