The majority of Golgi glycosyltransferases are type II membrane proteins with a small cytosolic tail at their N-terminus. Several mechanisms for localizing these glycosyltransferases to the Golgi have been proposed. InSaccharomyces cerevisiae, the phosphatidylinositol-4-phosphate-binding proteinScVps74p interacts with the cytosolic tail of a Golgi glycosyltransferase and contributes to its localization. In this study, we investigated whether a similar mechanism functions in the fission yeastSchizosaccharomyces pombe. First, we identifiedgpp74(+)(GPP34 domain-containing Vps74 homolog protein), a gene encoding theS. pombehomolog ofS. cerevisiaeVps74p. Deletion of thegpp74(+)gene resulted in the missorting of three Golgi glycosyltransferases,SpOch1p,SpMnn9p, andSpOmh1p, to vacuoles, but notSpAnp1p, indicating Gpp74p is required for targeting some glycosyltransferases to the Golgi apparatus. Gpp74p with an N-terminal GFP-tag localized to both the Golgi apparatus and the cytosol. Golgi localization of Gpp74p was dependent on the phosphatidylinositol 4-kinaseSpPik1p. Site-directed mutagenesis of hydrophobic and basic amino acids in the cytosolic tails ofSpOch1p andSpMnn9p resulted in their missorting to vacuoles, indicating these cytosolic N-terminal residues are important for localization in the Golgi. Unexpectedly, no prominent alternations in protein glycosylation were observed inS. pombe gpp74 Delta cells, probably due to the residual Golgi localization of someSpOch1p andSpMnn9p in these cells. Collectively, these results demonstrate that both Gpp74p-dependent and Gpp74p-independent mechanisms are responsible for the Golgi localization of glycosyltransferases to the Golgi inS. pombe.