This chapter focuses on protein sorting in the secretory pathway. From primary and secondary biosynthetic sites in the cytosol and mitochondrial matrix, respectively, proteins and lipids are distributed to more than 30 final destinations in membranes or membrane-bound spaces, where they carry out their programmed function. Molecular sorting is defined, in its most general sense, as the sum of the mechanisms that determine the distribution of a given molecule from its site of synthesis to its site of function in the cell. The final site of residence of a protein in a eukaryotic cell is determined by a combination of various factors, acting in concert: (1) site of synthesis, (2) sorting signals or zip codes, (3) signal recognition or decoding mechanisms, (4) cotranslational or posttranslational mechanisms for translocation across membranes, (5) specific fusion–fission interactions between intracellular vesicular compartments, and (6) restrictions to the lateral mobility in the plane of the bilayer. Improvements in cell fractionation, protein separation, and immune precipitation procedures in the past decade have made them possible. Very little is known about the mechanisms that mediate the localization and concentration of specific proteins and lipids within organelles. Various experimental model systems have become available for their study. The advent of recombinant DNA technology has shortened the time needed for obtaining the primary structure of proteins to a few months.