Two distinct proteins inhibiting phospholipase D (PLD) activity in rat brain cytosol were previously purified and identified as synaptojanin and AP180, which are specific to nerve terminals and associate with the clathrin coat. Two additional PLD-inhibitory proteins have now been purified and identified as the amphiphysins I and II, which forms a heterodimer that also associates with the clathrin coat. Bacterially expressed recombinant amphiphysins inhibited both PLD1 and PLD2 isozymes in vitro with a potency similar to that of brain amphiphysin (median inhibitory concentration of approximately 15 nm). Expressions of either amphiphysin in COS-7 cells reduced activity of endogenous PLD as well as exogenously expressed PLD1 and PLD2. Coprecipitation experiments suggested that the inhibitory effect of amphiphysins results from their direct interaction with PLDs. The NH(2) terminus of amphiphysin I was critical for both inhibition of and binding to PLD. Phosphatidic acid formed by signal-induced PLD is thought to be required for the assembly of clathrin-coated vesicles during endocytosis. Thus, the inhibition of PLD by amphiphysins, synaptojanin, and AP180 might play an important role in synaptic vesicle trafficking.