The beta 1,6 N-acetylglucosaminyltransferase (C2GnT) has been recently mapped to the cis/medial-Golgi compartment. To analyze the Golgi-targeting determinants of C2GnT, we constructed various deletion mutants of the enzyme fused to the enhanced green fluorescent protein (EGFP) and localized these proteins by fluorescence microscopy in living cells. We found that the N-terminal peptide encompassing amino acids 1 to 32 represents the minimal Golgi-targeting signal sufficient to localize EGFP to the same compartment as the full-length C2GnT. This peptide makes up the cytoplasmic and the transmembrane domains of the enzyme and was referred to as CTd (cytoplasmic and transmembrane domains). We compared the Golgi-targeting efficiency of the C2GnT-derived CTd with its homologous domains from other glycosyltransferases, including the H-type alpha(1,2)-fucosyltransferase (FucTI), the polypeptide N-acetylgalactosaminyltransferase-I (GalNAcT-I), the alpha(1,3)-fucosyltransferase VII (FucTVII), and the alpha(2,6)-sialyltransferase (ST6Gal-I) and found that the Golgi-targeting determinants of these glycosyltransferases were also composed of their cytosolic and transmembrane domains. To investigate whether the CTd of C2GnT could serve as a cis to medial Golgi-specific signal, we tested its ability to mislocalize two late-Golgi acting glycosyltransferases FucTI and FucTVII. We show that fusing the C2GnT-derived CTd with the catalytic domain of FucTVII resulted in a complete mislocalization of the enzyme to the C2GnT compartment, with a parallel alteration of sialyl-Lewis x synthesis and P-selectin binding. The intracellular distribution and activity of FucTI, however, were not affected. Thus, CTds of either early or late-Golgi acting glycosyltransferases represent the Golgi-targeting domains of these enzymes. In addition, we show that C2GnT-derived CTd can function as a cis/medial Golgi-targeting determinant.