Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport. These GTPases apparently function during the targeting of vesicles to the acceptor compartment. Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively. Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p. These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p. Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both. These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions. Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting. The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells. However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures. The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles. Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the trans-Golgi compartment, between the reactions regulated by Ypt1p and Sec4p. This step might involve budding of vesicles from the trans-Golgi. Alternatively, Ypt31/32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.