Arfaptin 1 is an ARF binding protein that inhibits PLD activation, vesicular trafficking and secretion. In the present report, we show that arfaptin 1 interacts with 'high speed' membranes independently of ARF. However, addition of myristoylated ARF3 (myrARF3) increases the association of arfaptin 1 with the membranes, suggesting that arfaptin 1 and ARF form a complex on the Golgi. Utilizing several deletion mutants of arfaptin 1 it is shown that the association of arfaptin 1 with myrARF3 is mediated via two binding sites on arfaptin 1. These two domains are needed for arfaptin 1 inhibition of PLD activation by myrARF3 in vitro.