The Sec1/Munc18 (SM) proteins constitute a conserved family with essential functions in SNAREmediated membrane fusion. Recently, a new protein-protein interaction site in Sec1p, designated the groove, was proposed. Here we show that a sec1 groove mutant yeast strain, sec1(w24), displays temperature sensitive growth and secretion defects. The yeast Sec1p and mammalian Munc18-1 grooves were shown to play an important role in the interaction with the SNAREs Sec9p and SNAP-25b, respectively. Incubation of SNAP-25b with Munc18-1 groove mutant resulted in a lag in the kinetics of SNARE complex assembly in vitro as compared to wild-type Munc18-1. The SNARE regulator SRO7 was identified as a multicopy suppressor of sec1(w24) groove mutant and an intact Sec1p groove was required for the plasma membrane targeting of Sro7p-SNARE complexes. Simultaneous inactivation of Sec1p groove and SRO7 resulted in reduced levels of exocytic SNARE complexes. Our results identify the groove as a conserved interaction surface in SM proteins. The results indicate that this structural element is important for interactions with Sec9p/SNAP-25 and participates, in concert with Sro7p, in the initial steps of SNARE complex assembly.