Intracellular vesicle targeting involves the interaction of vesicle proteins, termed v-SNAREs, with target membrane proteins, termed t-SNAREs. Assembly of v-SNARE–t-SNARE targeting complexes is modulated by members of the Sec1-Sly1 protein family, and by small guanosine triphosphatases termed Rabs. The interactions of these proteins during assembly of the endoplasmic reticulum–to-Golgi targeting complex in Saccharomyces cerevisiae were studied. The data suggest that the Rab protein Ypt1p transiently interacts with the t-SNARE Sed5p and results in displacement of the negative regulator Sly1p, allowing subsequent formation of the v-SNARE–t-SNARE targeting complex.