In eukaryotic cells, monomeric GTPases of the Ypt/Rab family function as regulators at defined steps of vesicular transport in exo‐ and endocytosis. Here we report on the isolation and characterization of two genes (YPT31 and YPT32) of the yeast Saccharomyces cerevisiae which encode members of the Ypt family exhibiting >80% sequence identity. Whereas the disruption of one of the two genes was phenotypically neutral, the disruption of both YPT31 and YPT32 led to lethality. Depletion of wild‐type Ypt31p or of a short‐lived ubiquitin‐Ypt31p in a ypt32 null background led to a massive accumulation of Golgi‐like membranes, an inhibition of invertase secretion and defects in vacuolar protein maturation. Similar alterations were observed in a conditional‐lethal ypt31–1 mutant at 30 min after shift to the non‐permissive temperature. According to subcellular fractionation, a significant part of Ypt31p appeared to be located in Golgi‐enriched membrane fractions. In accordance with this, indirect immunofluorescence using affinity‐purified anti‐Ypt31p antibodies gave a punctate staining similar to that observed with Golgi‐located proteins. From the phenotypic alterations observed in ypt31 and ypt32 mutants, it seems likely that the two GTPases are involved in intra‐Golgi transport or in the formation of transport vesicles at the most distal Golgi compartment.