Asparagine-linked glycosylation is the most ubiquitous protein co-translational modification in the endoplasmic reticulum (ER). The enzyme that catalyzes this process is called oligosaccharyl transferase (OT). It catalyzes the transfer of an oligosaccharyl moiety (Glc3Man9GlcNAc2) from the dolichol-linked pyrophosphate donor to the side chain of Asn within a consensus sequence of Asn-X-Thr/Ser, where X can be any amino acid residue except for Pro (1–3). This modification serves as a primary determinant for specific molecular recognition as well as protein folding and stability (4, 5) and therefore is an essential and highly conserved protein modification pathway in eukaryotic cells. It has been established in both yeast and higher eukaryotic organisms that this enzyme exists as a heteromeric, multisubunit complex in the ER membrane. In the last decade, because of the facility of yeast genetics, genes encoding 9 OT subunits in Saccharomyces cerevisiae have been cloned and identified (for review see Ref. 2 and earlier studies cited therein). Among them, the five genes encoding Ost1p, Ost2p, Stt3p, Wbp1p, and Swplp are essential for the viability of the cell; the OST4 gene is essential for growth of the cell at 37 °C but not at 25 °C; Ost3p, Ost5p, and Ost6p subunits are not essential for the viability of the yeast cell but are required for maximal enzyme activity. At present, many mammalian OT subunit proteins have also been identified, and some of them share high sequence identity and similarity with yeast homologs. Four of them were isolated from highly purified and active enzyme fractions: ribophorin I (homolog of yeast Ost1p), ribophorin II (homolog of yeast Swp1p), OST48 (homolog of yeast Wbp1p), and DAD1 (homolog of yeast Ost2p) (6–10). Recently STT3-A and STT3-B (homologs of yeast Stt3p), N33 and implantation-associated protein (homologs of yeast Ost3p and Ost6p, respectively), as well as OST4 (homolog of yeast Ost4p) have been characterized by genome-wide searches (11). These proteins have been demonstrated to be assembled together with ribophorin I, ribophorin II, OST48, and DAD1 into a multimeric complex similar to the yeast OT (11). Although genetic studies have yielded considerable information on this enzyme complex, one of the fundamental questions, the enzymatic mechanism of N-glycosylation, has remained unanswered. Specifically, the substrate recognition and/or catalytic sites, the role of each of the subunits, and how they interact structurally has continued to be obscure. In this review, we focus on explorations developed within the past 5 years to clarify the mechanism of this highly conserved protein modification pathway as well as the function of each of the subunits.