UDP-GlcNAc:alpha 3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I (GnTI) is an N(in)/C(out) (type II) membrane protein, localized in the medial-Golgi, that initiates the conversion of high mannose N-glycans to complex N-glycans. Anti-rabbit GnTI antibodies were generated using a purified, enzymatically active, bacterial recombinant fusion protein as immunogen. Rabbit GnTI was effectively retained in the Golgi complex of transfected COS-1 cells and murine L cells, as assessed by indirect immunofluorescence using the species-specific anti-GnTI antibodies; no surface expression of rabbit GnTI could be detected in the transfected cells. Rabbit GnTI, stably expressed in murine L cells, was localized by immunoperoxidase electron microscopy to the medial-cisternae of the Golgi stack. The role of the transmembrane domain of GnTI in Golgi localization was examined by generation of a hybrid construct containing the amino-terminal 31 amino acids of GnTI, corresponding to the 25-residue transmembrane (signal/anchor) domain and flanking hydrophilic sequences, fused with ovalbumin; this ovalbumin/GnTI hybrid molecule was retained in the Golgi complex of transfected COS cells and stably transfected murine L cells. No surface expression of ovalbumin/GnTI was detected. In contrast, ovalbumin fused to the equivalent domains of the human transferrin receptor, a type II cell-surface protein, was efficiently expressed on the cell surface of transfected cells. The ovalbumin/GnTI hybrid molecules in the transfected L cells were N-glycosylated, indicating an N(in)/C(out) membrane orientation, and were localized by immunoperoxidase electron microscopy to one or two cisternae of the medial-Golgi (90% of stained Golgi profiles showed medial-cisternae staining). These results show that a signal contained within the transmembrane domain and flanking residues of GnTI specifies medial-Golgi localization.