The Golgi lumenal GDPase plays an important role in the mannosylation of proteins and lipids of Saccharomyces cerevisiae by regulating the amount of GDP-mannose available in the Golgi lumen. The enzyme makes available GMP as an antiporter to be coupled with entry of GDP-mannose into the Golgi lumen from the cytosol. Using radiation inactivation and target analysis, we have now determined the functional molecular mass of the GDPase within the Golgi membrane and whether or not the enzyme has functional associations with other Golgi membrane proteins, including mannosyltransferases and the GDP-mannose transporter. The functional size of the GDPase was found to be approximately twice the estimated structural target size of the protein; this strongly suggests that the GDPase protein in situ functions as homodimer and does not require association with other membrane proteins for its function.