The involvement of the small GTP-binding protein ADP-ribosylation factor (ARF) in guanosine 5'- [y-thio] triphosphate (GTP[S])-dependent activation of phospholipase D (PLD) in HL-60 cells has been well established in vitro.In this study, we tested the effect of brefeldin A, which prevents ARF activation by inhibiting guanine-nucleotide-exchange activity, on PLD stimulation by receptor agonists (formylMet-Leu-Phe and ATP) and by the phorbol ester phorbol 12-myristate 13-acetate (PMA) in differentiated HL-60 cells. However, brefeldin A did not affect the activation of PLD at a time (1 h) when it eliminated the activity of the trans-Golgi enzyme galactosyltransferase. It also did not inhibit PLD activity in Golgienriched membranes treated with GTP[S] with or without ARF in vitro. However, longer times of brefeldin A treatment (> 6 h), progressively and completely inhibited the activation of PLD by formyl-MetLeu-Phe and partly inhibited (~5 0 % ) the activation by PMA. In contrast, long-term brefeldin A treatment did not inhibit the effect of GTP[S] on PLD in permeabilized HL-60 cells. Long-term brefeldin A treatment completely inhibited inositol phosphate production in response to formyl-Met-Leu-Phe and ATP, indicating that it affected inositolphospholipid-specific phospholipase C activity. These data indicate that the rapid inhibitory effect of brefeldin A on Golgi function is not associated with inhibition of receptormediated or PMA-mediated PLD activation in HL-60 cells. However, longer-term effects, presumably arising from the disruption of the Golgi, lead to a total inhibition of agonist activation of PLD and inositolphospholipid-specific phospholipase C. In summary, these results do not support a role for brefeldin-A-sensitive ARF in agonist regulation of PLD in HL-60 cells.Keywords: phospholipase D ; ADP-ribosylation factor; brefeldin A; inositolphospholipid; Golgi.Brefeldin A is a fungal metabolite, initially reported to inhibit protein secretion [l]. It inhibits vesicular transport in the Golgi complex and causes a rapid resorption of most of the Golgi membrane into the endoplasmic reticulum [2] and association of Golgi enzymes with the endoplasmic reticulum, thereby eliminating the Golgi complex as a morphologically distinct organelle [3]. Morphological changes in the Golgi and redistribution of Golgi markers are observed within minutes of addition of brefeldin A. Upon removal of brefeldin A, the morphology of the Golgi is rapidly restored, and normal protein transport and sorting resumes.The earliest detectable event in the action of brefeldin A is prevention of the GTP-dependent interaction with Golgi membranes of ADP-ribosylation factor (ARF), a small GTP-binding protein [4]. A protein that catalyzes the exchange of guanine nucleotide on ARF has been identified in Golgi preparations and shown to be specifically inhibited by brefeldin A [5, 61. Since ARF binding on Golgi membranes is required for the association of the p-coatamer protein (/I-COP), a component of coatomers, PLD activation by guanosine 5...