Mammalian phospholipase D (PLD) has been reported to be a key enzyme for epidermal growth factor (EGF)-induced cellular signaling, however, the regulatory mechanism of PLD is still unclear. In this report, we found that Munc-18-1 is a potent negative regulator of PLD in the basal state and that its inhibition is abolished by EGF stimulation. We investigated PLD-binding proteins obtained from rat brain extract, and identified a 67-kDa protein as Munc-18-1 by peptide-mass finger-printing. The direct association between PLD and Munc-18-1 was confirmed by in vitro binding analysis using the purified proteins, and their binding sites were identified as the phox homology domain of PLD and multiple sites of Munc-18-1. PLD activity was potently inhibited by Munc-18-1 in vitro (IC50 = 2-5 nm), and the cotransfection of COS-7 cells with Munc-18-1 and PLD inhibited basal PLD activity in vivo. In the basal state, Munc-18-1 coprecipitated with PLD and colocalized with PLD2 at the plasma membrane of COS-7 cells. EGF treatment triggered the dissociation of Munc-18-1 from PLD when PLD was activated by EGF. The dissociation of the endogenous interaction between Munc-18-1 and PLD, and the activation of PLD by EGF were also observed in primary cultured chromaffin cells. These results suggest that Munc-18-1 is a potent negative regulator of basal PLD activity and that EGF stimulation abolishes this interaction.