Rat liver microsomal glycoproteins were purified by affinity chromatography on concanavalin A Sepharose columns from membrane and content fractions, separated from rough microsomes (RM) treated with low concentrations of deoxycholate (DOC). All periodic acid-Schiff (PAS)-positive glycoproteins of RM showed affinity for concanavalin A Sepharose; even after sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, most of the microsomal glycoproteins bound [125I]concanavalin A added to the gels, as detected by autoradiography. Two distinct sets of glycoproteins are present in the membrane and content fractions derived from RM. SDS acrylamide gel electrophoresis showed that RM membranes contain 15--20 glycoproteins (15--22% of the total microsomal protein) which range in apparent mol wt from 23,000 to 240,000 daltons. A smaller set of glycoproteins (five to seven polypeptides), with apparent mol wt between 60,000 and 200,000 daltons, was present in the microsomal content fraction. The disposition of the membrane glycoproteins with respect to the membrane plane was determined by selective iodination with the lactoperoxidase (LPO) technique. Intact RM were labeled on their outer face with 131I and, after opening of the vesicles with 0.05% DOC, in both faces with 125I. An analysis of iodination ratios for individual proteins separated electrophoretically showed that in most membrane glycoproteins, tyrosine residues are predominantly exposed on the luminal face of the vesicles, which is the same face on which the carbohydrate moieties are exposed. Several membrane glycoproteins are also exposed on the cytoplasmic surface and therefore have a transmembrane disposition. In this study, ribophorins I and II, two integral membrane proteins (mol wt 65,000 and 63,000) characteristic of RM, were found to be transmembrane glycoproteins. It is suggested that the transmembrane disposition of the ribophorins may be related to their possible role in ribosome binding and in the vectorial transfer of nascent polypeptides into the microsomal lumen.