Canine pancreas rough microsomes were solubilized in a high salt buffer containing sodium cholate, a detergent extract prepared by high speed centrifugation, and vesicles were reconstituted from the extract by a detergent dialysis procedure. The reconstituted vesicles, lacking resident lumenal proteins, translocated in vitro-synthesized preprolactin in a cotranslational, SRP-dependent manner. The translocated precursor was processed to mature prolactin and protected from digestion by exogenous protease. Vesicles were also reconstituted from detergent extracts depleted of glycoproteins by chromatography on concanavalin-Sepharose. The depleted vesicles, containing a full complement of SRP receptor but deficient in the glycoprotein subunit of signal peptidase, the ribophorins, and other glycoprotein components, were functional in the targeting and binding of nascent preprolactin but exhibited a greatly diminished capacity for translocation.