Eukaryotic cells are characterized by the existence of membrane-bound subcellular compartments which perform a variety of specialized functions. Proteins destined for these compartments begin their synthesis in the cytosol and must be subsequently targeted to their functional compartment by specific signal sequences present in the newly synthesized polypeptide chain. The translocation of preproteins across biological membranes is a fundamental process of intracellular trafficking and organelle biogenesis. Entry into the secretory pathway occurs by translocation of proteins into or across the membrane of the endoplasmic reticulum (ER). This process involves two distinct steps which are dependent on the orchestrated action of several proteins. The initial step of targeting involves recognition of the signal sequence and delivery of the protein precursor to the ER in a translocation competent conformation. The subsequent translocation event is characterized by interaction of the preprotein with the translocation channel followed by unidirectional movement across the lipid bilayer of the ER membrane into the lumenal space. The study of the mechanism of the translocation process is one of the most intriguing and rapidly advancing areas in cell biology. Here we review recent findings in both the yeast Saccharomyces cerevisiae and mammals concerning the mechanisms of the translocation step and discuss the roles of the proteins implicated in this process.