Glc3Man9GlcNAc2-P-P-Dol serves as the major precursor for the biosynthesis of asparagine-linked glycoproteins in eukaryotes. The first 5 of the 9 mannosyl residues during the assembly of the oligosaccharide moiety within the dolichol cycle in the endoplasmic reticulum are incorporated directly by the action of GDP-Man-requiring mannosyltransferases while the remaining last 4 mannosyl residues are transferred by Man-P-Dol-requiring enzymes. In an earlier study (Shailubhai, K., Illeperuma, C., Tayal, M., and Vijay, I. K. (1990) J. Biol. Chem. 265, 14105-14108), we identified the enzyme UDP-Glc:Dol-P glucosyltransferase by photolabeling rat mammary microsomes with 5-N3-[beta-32P]UDP-Glc. Applying a similar strategy, GDP-hexanolamine-125I-azidosalicylic acid, an analog of GDP-Man, was found to photolabel two polypeptides of 37 and 69 kDa among the microsomal proteins of the rat mammary gland. A differential ammonium sulfate saturation (60-80%) of the detergent-solubilized microsomal proteins enriched the 69-kDa polypeptide. Photolabeling of this polypeptide was specifically inhibited by guanine-containing nucleotides and nucleotide-sugars and was associated with a GDP-Man-requiring mannosyltransferase. The mannosyltransferase was purified nearly 16,000-fold and shown to contain the 69-kDa polypeptide. The purified enzyme catalyzes the transfer of [14C]Man from GDP-[14C]Man to Man beta 1-->4GlcNAc beta 1-->4GlcNAc-P-P-Dol in alpha 1,3-linkage to give [14C]Man alpha 1-->3Man beta 1-->4GlcNAc beta 1-->4GlcNAc-P-P-Dol as the product. Antibodies raised against the 69-kDa polypeptide removed the enzymatic activity from the detergent extract of the rat mammary microsomes and reacted specifically with a polypeptide band of the same size on immunoblots. The purified enzyme showed a pH optima of 7.4-7.8, Km approximately 4 microM for GDP-Man, approximately 2-fold activation by phosphatidylcholine, and a strong inhibition by sulfhydryl-selective reagents, N-ethylmaleimide and p-chloromercuribenzoate. The availability of the highly purified enzyme and a monospecific antibody should allow its molecular cloning for investigating the regulation of the machinery for protein N-glycosylation upon hormonally modulated growth and differentiation of the mammary gland during its ontogeny.