ARFs constitute a family of structurally related proteins that forms a subset of the ras GTPases. In chromaffin cells, secretagogue-evoked stimulation triggers the rapid translocation of ARF6 from secretory granules to the plasma membrane and the concomitant activation of PLD in the plasma membrane. Both PLD activation and catecholamine secretion are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6. ARNO, a potential guanine nucleotide exchange factor for ARF6, is expressed and localized in the plasma membrane of chromaffin cells. Using permeabilized cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits both PLD activation and catecholamine secretion. Chromaffin cells express PLD1 at the plasma membrane. We found that microinjection of the catalytically inactive PLD1(K898R) dramatically reduces catecholamine secretion monitored by amperometry, most likely by interfering with a late postdocking step of calcium-regulated exocytosis. We propose that ARNO-ARF6 participate in the exocytotic reaction by controlling the plasma membrane-bound PLD1. By generating fusogenic lipids at the exocytotic sites, PLD1 may represent an essential component of the fusion machinery in neuroendocrine cells.