The Saccharomyces cerevisiae Golgi lumenal guanosine diphosphatase is hypothesized to generate GMP which in turn allows entry of GDP-mannose into the lumen to serve as substrate for mannosylation of proteins and lipids. We have recently shown in studies in vivo that this GDPase is required for protein and sphingolipid mannosylation in the Golgi lumen of S. cerevisiae. We have now isolated Golgi-vesicles from wild type and gda1 null mutants (GDPase defective) and have found that the initial rate of GDP-mannose entry into mutant vesicles was 5-fold lower than into those of wild type. Because the concentration of GDP within vesicles is insufficient to inhibit Golgi lumenal mannosyltransferases and the null mutant vesicles are impaired in synthesis of Golgi mannoproteins, the above results demonstrate that the reduced availability of GDP-mannose in the null mutants is the cause for altered Golgi mannosylation of macromolecules.