SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) are cytoplasmically oriented membrane proteins that reside on vesicular carriers (v-SNARE) and target organelles (t-SNARE). The pairing of a stage-specific v-SNARE with its cognate t-SNARE may mediate the specificity of membrane traffic. In the yeast Saccharomyces cerevisiae transport between the endoplasmic reticulum and Golgi complex employs two v-SNAREs, Bos1p and Sec22p, each containing a domain that is related to the neuronal v-SNARE synaptobrevin. Sed5p, which is homologous to syntaxin, is the t-SNARE that functions at this stage of the secretory pathway. Here we report that regions of Bos1p and Sec22p, which are homologous to synaptobrevin, bind to the syntaxin-like domain of Sed5p. Furthermore, we demonstrate that efficient v-SNARE/t-SNARE interactions require the participation of both v-SNAREs, indicating that, unlike post-Golgi membrane traffic, the active form of the endoplasmic reticulum to Golgi v-SNARE is a heteromeric complex.