Members of the Ras superfamily of GTP-binding proteins are involved in a variety of cellular processes, including signal transduction, cytoskeletal organization and protein transport. GTP-binding proteins of the Ypt/Rab family direct vesicular protein transport in the secretory and endocytic pathways in the yeast Saccharomyces cerevisiae (Ypt proteins) and in mammalian systems (Rab proteins). The cellular activity of monomeric GTP-binding proteins is influenced by proteins that regulate GDP/GTP exchange and GTP hydrolysis. GTPase-activating proteins (GAPs) can increase the slow intrinsic GTPase activity of GTP-binding proteins by several orders of magnitude. As GAPs modulate the activity of GTP-binding proteins, they are thought to give a biochemical handle on the functioning of Ypt/Rab proteins in transport vesicle budding and docking or fusion at donor and acceptor membranes. We report here the first cloned GTPase-activating protein for the Ypt/Rab protein family. The gene, GYP6 (GAP of Ypt6 protein), encodes a protein of 458 amino acids which is highly specific for the Ypt6 protein and shows little or no cross-reactivity with other Ypt/Rab family members or with H-Ras p21.