We have isolated the gdp1+ gene from Schizosaccharomyces pombe coding for a membrane protein with guanosine diphosphatase (GDPase) activity, which is highly homologous to Golgi GDPases isolated from other yeast species. The gdp1+ product, Gdp1p, displays both GDPase and uridine diphosphatase (UDPase) activities in vitro, with a strong dependence for calcium and manganese cations. The observation of a defect in N-glycosylation of invertase in S. pombe Deltagdp1 cells together with the ability of gdp1+ to functionally complement the defective O-mannosylation of chitinase in Saccharomyces cerevisiae cells disrupted in the GDA1 gene (gdp1+ homolog), suggests a main role for Gdp1p in protein glycosylation in fission yeast.